U.S. patent application number 12/621686 was filed with the patent office on 2010-05-20 for pluripotent stem cell culture on micro-carriers.
Invention is credited to Shelley Nelson.
Application Number | 20100124781 12/621686 |
Document ID | / |
Family ID | 41820296 |
Filed Date | 2010-05-20 |
United States Patent
Application |
20100124781 |
Kind Code |
A1 |
Nelson; Shelley |
May 20, 2010 |
Pluripotent Stem Cell Culture on Micro-Carriers
Abstract
The present invention is directed to methods for the growth,
expansion and differentiation of pluripotent stem cells on
micro-carriers.
Inventors: |
Nelson; Shelley; (Skillman,
NJ) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
41820296 |
Appl. No.: |
12/621686 |
Filed: |
November 19, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61116447 |
Nov 20, 2008 |
|
|
|
Current U.S.
Class: |
435/366 ;
435/377 |
Current CPC
Class: |
C12N 5/0075 20130101;
C12N 5/0602 20130101; C12N 5/0676 20130101; C12N 5/0606 20130101;
C12N 2501/70 20130101; C12N 2501/999 20130101; C12N 2509/00
20130101; C12N 2531/00 20130101; C12N 2506/02 20130101 |
Class at
Publication: |
435/366 ;
435/377 |
International
Class: |
C12N 5/071 20100101
C12N005/071 |
Claims
1. A method for the propagation of pluripotent stem cells,
comprising the steps of: a. Attaching a population of pluripotent
stem cells to a first volume of micro-carriers, b. Culturing the
pluripotent stem cells on the first volume of micro-carriers, c.
Removing the pluripotent stem cells from the first volume of
micro-carriers, and d. Attaching the population of pluripotent stem
cells to a second volume of micro-carriers.
2. The method of claim 1, wherein the steps of culturing, removing
and attaching the pluripotent stem cells on micro-carriers are is
repeated using subsequent volumes of micro-carriers.
3. The method of claim 1, wherein the first volume of
micro-carriers is selected from the group consisting of dextran
micro-carriers and polystyrene micro-carriers.
4. The method of claim 1, wherein the second volume of
micro-carriers is selected from the group consisting of dextran
micro-carriers and polystyrene micro-carriers.
5. The method of claim 1, wherein the pluripotent stem cells are
attached to the first volume of micro-carriers in medium containing
a Rho kinase inhibitor.
6. The method of claim 1, wherein the pluripotent stem cells are
attached to the second volume of micro-carriers in medium
containing a Rho kinase inhibitor.
7. The method of claim 1, wherein the pluripotent stem cells are
removed from the first volume of micro-carriers by enzymatic
treatment.
8. The method of claim 1, wherein the pluripotent stem cells are
removed from the second volume of micro-carriers by enzymatic
treatment.
9. The method of claim 1, wherein the first volume of
micro-carriers is removed from the pluripotent stem cells prior to
attaching the cells to the second volume of micro-carriers.
10. A method to differentiate pluripotent stem cells to
insulin-expressing cells on micro-carriers comprising the steps of:
a. Attaching the pluripotent stem cells to a volume of
micro-carriers, b. Differentiating the pluripotent stem cells to
cells expressing markers characteristic of the definitive endoderm
lineage, c. Differentiating the cells expressing markers
characteristic of the definitive endoderm lineage into cells
expressing markers characteristic of the pancreatic endoderm
lineage, d. Differentiating the cells expressing markers
characteristic of the pancreatic endoderm lineage into cells
expressing markers characteristic of the pancreatic endocrine
lineage, and e. Differentiating the cells expressing markers
characteristic of the pancreatic endocrine lineage into
insulin-expressing cells.
Description
[0001] The present invention claims priority to application Ser.
No. 61/116,447, filed Nov. 20, 2008.
FIELD OF THE INVENTION
[0002] The present invention is directed to methods for the growth,
expansion and differentiation of pluripotent stem cells on
micro-carriers.
BACKGROUND
[0003] Pluripotent stem cells, such as, for example, embryonic stem
cells have the ability to differentiate into all adult cell types.
As such, embryonic stem cells may be a source of replacement cells
and tissue for organs that have been damaged as a result of
disease, infection, or congenital abnormalities. The potential for
embryonic stem cells to be employed as a replacement cell source is
hampered by the difficulty of propagating the cells in vitro while
maintaining their pluripotency.
[0004] Current methods of culturing undifferentiated embryonic stem
cells require complex culture conditions, such as, for example,
culturing the embryonic stem cells in the presence of a feeder cell
layer. Alternatively, media obtained by exposure to feeder cell
cultures may be used to culture embryonic stem cells. Culture
systems that employ these methods often use cells obtained from a
different species than that of the stem cells being cultivated
(xenogeneic cells). Additionally, these culture systems may be
supplemented with animal serum.
[0005] Embryonic stem cells provide a potential resource for
research and drug screening. At present, large-scale culturing of
human embryonic stem cell lines is problematic and provides
substantial challenges. Current in vitro methods to propagate
pluripotent stem cells are carried out in tissue flasks on planar
surfaces pre-coated with extracellular matrix (ECM) proteins or
feeder cells. Planar cultures also require frequent subculturing
because their limited surface area cannot support long-term growth
of pluripotent stem cells. Micro-carrier-based methods of
pluripotent stem cell culture may provide a solution.
Micro-carriers have a high surface-area-to-volume ratio and,
therefore, eliminate the surface area restriction of growing
pluripotent stem cells on planar surfaces.
[0006] For example, Fok et al disclose stirred-suspension culture
systems for the propagation of undifferentiated ESC-micro-carrier
and aggregate cultures (Stem Cells 2005; 23:1333-1342.)
[0007] In another example, Abranches et al disclose the testing of
Cytodex 3.RTM. (GE Healthcare Life Sciences, NJ), a microporous
micro-carrier made up of a dextran matrix with a collagen layer at
the surface for its ability to support the expansion of the mouse
S25 ES cell line in spinner flasks (Biotechnol. Bioeng. 96 (2007),
pp. 1211-1221.)
[0008] In another example, US20070264713 disclose a process for
cultivating undifferentiated stem cells in suspension and in
particular to a method for cultivating stem cells on micro-carriers
in vessels.
[0009] In another example, WO2006137787 disclose a screening tool
is used which comprises particulate matter or micro-carriers, such
as beads, attached to a solid support, such as a micro titer plate,
for the cultivation of cells on said micro-carriers.
[0010] In another example, WO2008004990 disclose a method of
promoting the attachment, survival and/or proliferation of a stem
cell in culture, the method comprising culturing a stem cell on a
positively-charged support surface.
[0011] In another example, WO2007012144 disclose a bioreactor,
comprising: a support surface; and a synthetic attachment
polypeptide bound to the support surface wherein the synthetic
attachment polypeptide is characterized by a high binding affinity
for an embryonic stem cell or a multipotent cell.
SUMMARY
[0012] The present invention provides methods for the growth,
expansion and differentiation of pluripotent stem cells on
micro-carriers.
[0013] In one embodiment, the present invention provides a method
for the propagation of pluripotent stem cells, comprising the steps
of: [0014] a. Attaching a population of pluripotent stem cells to a
first volume of micro-carriers, [0015] b. Culturing the pluripotent
stem cells on the first volume of micro-carriers, [0016] c.
Removing the pluripotent stem cells from the first volume of
micro-carriers, and [0017] d. Attaching the population of
pluripotent stem cells to a second volume of micro-carriers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1: Rho-kinase inhibitor promotes attachment and growth
of human embryonic stem cells to micro-carriers. Images of H9 cells
grown in static culture for 2 days on HILLEX.RTM.II micro-carriers
(Solohill, MI). The cells were cultured in mouse embryonic
fibroblast conditioned medium (MEF-CM) with or without 10 .mu.M Rho
Kinase inhibitor, Y27632 ((Sigma-Aldrich, MO) A and B,
respectively).
[0019] FIG. 2: H9 cells grown on micro-carriers. H9 cells were
allowed to attach to various micro-carriers and placed on a rocking
platform at 37.degree. C. Plastic micro-carriers, ProNectinF
micro-carriers, HILLEX.RTM.II micro-carriers (Solohill, MI), and
Plastic Plus micro-carriers, were used (A, B, C, D respectively).
Growth after 3 days showed cells on HILLEX.RTM.II (Solohill, MI)
with best cell attachment to the micro-carriers. Arrows identify
cells forming aggregates without attachment to the
micro-carriers.
[0020] FIG. 3: H9 cell proliferation on micro-carriers. H9 cells
were attached to HILLEX.RTM.II micro-carriers, ProNectinF
micro-carriers, Plastic Plus micro-carriers, and Plastic
micro-carriers (Solohill, MI) and placed in a 6 well dish on a
rocking platform at 37.degree. C. in the presence of 10 .mu.M
Y27632 (Sigma-Aldrich, MO) and MEF-CM. The initial cell seeding
density is the value at day 0. Day 3 and day 5 cell numbers are
shown.
[0021] FIG. 4: H1 cell images after attachment to micro-carriers.
Images of cells at days 3, 5 and 7 are shown attached to ProNectinF
micro-carriers, Plastic Plus micro-carriers, and Plastic
micro-carriers. The cells were grown in MEF-CM with 10 .mu.M Y27632
(Sigma-Aldrich, MO) in a 12 well dish on a rocking platform at
37.degree. C. Cells formed aggregates independent of binding to
Plastic Plus and Plastic micro-carriers (arrows in G, H).
[0022] FIG. 5: H1 cell images after attachment to micro-carriers.
Images of cells at days 3, 5 and 7 are shown attached to Cytodex
1.RTM. micro-carriers, Cytodex 3.RTM. micro-carriers (GE Healthcare
Life Sciences, NJ) and HILLEX.RTM.II micro-carriers (Solohill, MI).
The cells were grown in MEF-CM with 10 .mu.M Y27632 (Sigma-Aldrich,
MO) in a 12 well dish on a rocking platform at 37.degree. C.
[0023] FIG. 6: H1 cell proliferation on micro-carriers. H1 cells
were allowed to attach to HILLEX.RTM.II micro-carriers (Solohill,
MI), Cytodex 1.RTM. micro-carriers (GE Healthcare Life Sciences,
NJ), Cytodex 3.RTM. micro-carriers (GE Healthcare Life Sciences,
NJ), ProNectinF micro-carriers (Solohill, MI), Plastic Plus
micro-carriers (Solohill, MI), and Plastic micro-carriers
(Solohill, MI) and placed in a 12 well dish on a rocking platform
at 37.degree. C. in the presence of 10 .mu.M Y27632 (Sigma-Aldrich,
MO) and MEF-CM. The initial cell seeding density is the value at
day 0. Day 3, 5, and 7 cell numbers are shown. The initial seeding
density was 13,333 cells/cm.sup.2, as indicated by the line.
[0024] FIG. 7: H9 cell proliferation on micro-carriers in various
concentrations of Rho kinase inhibitors. Cells were grown in a 12
well plate on a rocking platform and counted at day 4 and 7 to
determine attachment and proliferation rate. A. H9 cells were grown
in MEF-CM with 1, 2.5, 5, or 10 .mu.M Y27632 (Sigma-Aldrich, MO).
B. H9 cells were grown in MEF-CM with 0.5, 1, 2.5, or 5 .mu.M
Glycyl-H 1152 dihydrochloride (Tocris, MO).
[0025] FIG. 8: H1 cells were grown in decreasing concentrations of
Rho kinase inhibitors. H1p38 cells were grown in the presence of
Y27632 (Sigma-Aldrich, MO) or Glycyl-H 1152 dihydrochloride
(Tocris, MO) for two days at decreasing concentrations (10 .mu.M/5
.mu.M, 2.5 .mu.M/0.5 .mu.M or 1.0 .mu.M/0.5 .mu.M) or at 0.25 .mu.M
Glycyl-H 1152 dihydrochloride (Tocris, MO) continuously. Cells were
allowed to attach to HILLEX.RTM.II (Solohill, MI), Cytodex 1.RTM.,
or Cytodex 3.RTM. ((GE Healthcare Life Sciences, NJ) A, B, C,
respectively). Cells were counted at 3, 5 and 7 days post
seeding.
[0026] FIG. 9: Determination of cell attachment to micro-carriers
at different seeding densities in spinner flasks. H1 cells were
seeded onto Cytodex 3.RTM. (GE Healthcare Life Sciences, NJ)
micro-carriers at the densities listed on the left; Low
(0.4.times.10.sup.4 cells/cm.sup.2), Mid (1.2.times.10.sup.4
cells/cm.sup.2) or High (3.times.10.sup.4 cells/cm.sup.2). At 3, 5
and 7 days the cells were imaged and the percentage of
micro-carriers with cells attached was determined (embedded in
image).
[0027] FIG. 10: Cell growth on micro-carriers in spinner flasks is
affected by the initial seeding densities. H1 cells were seeded
onto Cytodex 3.RTM. (GE Healthcare Life Sciences, NJ)
micro-carriers at the densities listed on the left; Low
(0.4.times.10.sup.4 cells/cm.sup.2), Mid (1.2.times.10.sup.4
cells/cm.sup.2) or High (3.times.10.sup.4 cells/cm.sup.2). At 3, 5
and 7 days the cells were dissociated from the micro-carriers and
counted.
[0028] FIG. 11: Determination of cell growth rate on micro-carriers
at different seeding densities in spinner flasks. H1 cells were
seeded onto Cytodex 3.RTM. (GE Healthcare Life Sciences, NJ)
micro-carriers at different densities (day 0); Low
(0.4.times.10.sup.4 cells/cm.sup.2), Mid (1.2.times.10.sup.4
cells/cm.sup.2) or High (3.times.10.sup.4 cells/cm.sup.2). At 3, 5
and 7 days the cells were dissociated from the micro-carriers and
counted. The fold increase in cell number is shown versus initial
seeding density.
[0029] FIG. 12: H1 cells grown on Cytodex 3.RTM. micro-carriers (GE
Healthcare Life Sciences, NJ) were imaged after 7 days in culture.
The cells received MEF-CM without Rho kinase inhibitor from day 3
onward. The cells remained attached to the micro-carriers.
[0030] FIG. 13: H9 cells growth and dissociation of H9 cells on
HILLEX.RTM.II micro-carriers (Solohill, MI). A, B 10.times. and
20.times. images of H9 cells grown for 6 days on HILLEX.RTM.II
micro-carriers (Solohill, MI). C, 20.times. image of cells
dissociated from HILLEX.RTM.II micro-carriers (Solohill, MI) for 10
minutes with 0.05% Trypsin/EDTA. D, 20.times. image of cells
dissociated from HILLEX.RTM.II micro-carriers (Solohill, MI) for 10
minutes with TrypLE.TM. Express.
[0031] FIG. 14: Dissociation of H9 cells from micro-carriers. H9
cells grown on HILLEX.RTM.II (Solohill, MI) on a rocking platform,
were dissociated with TrypLE.TM. Express or 0.05% Trypsin/EDTA. The
number of cells and their viability is shown, A and B
respectively.
[0032] FIG. 15: Dissociation of H1 cells from micro-carriers. H1
cells grown on Cytodex 3.RTM.(GE Healthcare Life Sciences, NJ) in a
spinner flask were dissociated with TrypLE.TM. Express (Invitrogen,
CA), Accutase.TM. or Collagenase (10 mg/ml). The number of cells
and their viability is shown, A and B respectively.
[0033] FIG. 16: H9 cells grown on HILLEX.RTM.II (Solohill, MI)
micro-carriers do not transfer between micro-carriers.
[0034] FIG. 17: H9 at passage 43 were grown for 5 passages on
HILLEX.RTM.II (Solohill, MI) micro-carriers in a spinner flask.
Cells were counted every 2 to 3 days and passaged when cells
reached 1-2.times.10.sup.5 cells/cm.sup.2.
[0035] FIG. 18: H9 cells at passage 43 were grown for 5 passages on
Cytodex 3.RTM. micro-carriers (GE Healthcare Life Sciences, NJ) in
a spinner flask. Cells were counted every 2 to 3 days and passaged
when cells reached 1-2.times.10.sup.5 cells/cm.sup.2.
[0036] FIG. 19: Fluorescent-activated cell sorting (FACS) shows
pluripotency of H9 cells grown in spinner flasks. A, The majority
of H9 p43 cells grown on HILLEX.RTM.II (Solohill, MI)
micro-carriers express of pluripotency proteins. Passage 1 and 3
cells were not evaluated for TRA-1-81. B, The majority of H9 p43
cells grown on Cytodex 3.RTM. (GE Healthcare Life Sciences, NJ)
micro-carriers express of pluripotency proteins. Passage 1 cells
were not evaluated for TRA-1-81.
[0037] FIG. 20: H1 p49 cells were grown for 5 passages on Cytodex
1.RTM. micro-carriers (GE Healthcare Life Sciences, NJ) in a
spinner flask. Cells were counted every 2 to 3 days and passaged
when cells reached 4-8.times.10.sup.4 cells/cm.sup.2.
[0038] FIG. 21: H1 cells at passage 49 were grown for 5 passages on
Cytodex 3.RTM. micro-carriers (GE Healthcare Life Sciences, NJ) in
a spinner flask. Cells were counted every 2 to 3 days and passaged
when cells reached 1-2.times.10.sup.5 cells/cm.sup.2.
[0039] FIG. 22: Fluorescence activated cell sorting (FACS) shows
pluripotency of H1 cells grown in spinner flasks.
[0040] FIG. 23: Population doublings of H1 and H9 cells on
micro-carriers. Population doubling times were calculated from day
3 to the day of passaging (day 5, 6 or 7).
[0041] FIG. 24: H9 cells cultured on micro-carriers in defined
media. The cells were cultured on HILLEX.RTM.II (HII, (Solohill,
MI)) or Cytodex 3.RTM. (C3, (GE Healthcare Life Sciences, NJ)).
Cells were cultured on micro-carriers in one of the following
media; mTESR (StemCell Technologies, Vancouver, Canada), StemPro or
MEF-CM. 10 .mu.M Y27632 (Y, (Sigma-Aldrich, MO)) or 2.5 .mu.M
Glycyl-H 1152 dihydrochloride (H, (Tocris, MO)) was added to the
media. Growth rate at 3, 5 and 7 days post seeding was
determined.
[0042] FIG. 25: H1 cells at passage 38 were cultured on
micro-carriers in defined media. The cells were cultured on
HILLEX.RTM.II (HII, (Solohill, MI)) or Cytodex 3.RTM. (C3, (GE
Healthcare Life Sciences, NJ)) micro-carriers. Cells were cultured
on micro-carriers in one of the following medias; mTESR (StemCell
Technologies, Vancouver, Canada), StemPro and MEF-CM. 10 .mu.M
Y27632 (Y, (Sigma-Aldrich, MO)) or 2.5 .mu.M Glycyl-H 1152
dihydrochloride (H, (Tocris, MO)) was added to the media. Growth
rate at 3, 5 and 7 days post seeding was determined.
[0043] FIG. 26: H1 cells at passage 50 were cultured on
HILLEX.RTM.II (Solohill, MI)) micro-carriers with defined medium in
a spinner flask. A, Images of H1 p50 cells grown in MEF-CM after 3,
7, or 9 days in a spinner flask. B, Images of H1 p50 cells grown in
mTESR (StemCell Technologies, Vancouver, Canada) after 3, 7, or 9
days. Arrows identify cell clusters not attached to the
micro-carriers.
[0044] FIG. 27: Differentiation of human embryonic stem cells
passaged five times in spinner flasks. A, H9 cells at passage 43
were passaged five times on Cytodex 3.RTM. micro-carriers (GE
Healthcare Life Sciences, NJ). B, H1 cells at passage 49 were
passaged five times on Cytodex 1.RTM. micro-carriers (GE Healthcare
Life Sciences, NJ). Both cell types were released from the
micro-carriers and seeded onto MATRIGEL (BD Biosciences, CA) coated
plates. At 80-90% confluency the cells were exposed to a protocol
that is capable of differentiating embryonic stem cells to
definitive endoderm. The cells were then analyzed by FACS for the
percentage of cells expressing CXCR4, a definitive endoderm marker.
The percent of CXCR4 positive cells is in the upper right corner of
the plot.
[0045] FIG. 28: Differentiation of H1 cells on micro-carriers to
definitive endoderm. Here FACS plots display the percentage of
cells expressing the definitive endoderm marker CXCR4. Percent
positive is in the upper right corner. Cells were all expanded on
micro-carriers in spinner flasks prior to treatment. A, H1 cells at
passage 40 were grown on Cytodex 1.RTM. micro-carriers (GE
Healthcare Life Sciences, NJ) for 6 days after passage 5 prior to
differentiation. B, H1 cells at passage 40 were grown on Cytodex
3.RTM. micro-carriers (GE Healthcare Life Sciences, NJ) for 8 days
after passage 1 prior to differentiation. C, H1 cells at passage 50
were grown on HILLEX.RTM.II micro-carriers (Solohill, MI) for 6
days after passage 1 prior to differentiation.
[0046] FIG. 29: Differentiation of H1 cells on Cytodex 3.RTM.
micro-carriers (GE Healthcare Life Sciences, NJ) to definitive
endoderm. A, H1 cells at passage 40 were grown on micro-carriers
for eight days. B, H1 cells at passage 40 were grown on
micro-carriers for 11 days. Both cell population were then
differentiated to definitive endoderm on a rocking platform at
37.degree. C. Here FACS plots display the percentage of cells
expressing the definitive endoderm marker CXCR4. Percent positive
is in the upper right corner.
[0047] FIG. 30: Differentiation of cells of the human embryonic
stem cell line H1, cultured on micro-carriers to definitive
endoderm. FACS results for the percent positive CXCR4 cells are
shown on the Y-axis. H1 cells were grown on HILLEX.RTM.II, Cytodex
1.RTM. or Cytodex 3.RTM. micro-carriers prior to and during
differentiation.
[0048] FIG. 31: Differentiation of cells of the human embryonic
stem cell line H1 cultured on micro-carriers to pancreatic endoderm
cells. CT values are shown on the Y-axis for pancreatic endodermal
markers, Ngn3, NRx6.1 and Pdx1. H1 cells were differentiated on
HILLEX.RTM.II (HII), Cytodex 1.RTM.(C1) or Cytodex 3.RTM. (C3)
micro-carriers in either DMEM-High Glucose (HG) or DMEM-F12 (F12)
media. The differentiation protocol lasted 13 days.
[0049] FIG. 32: Differentiation of cells of the human embryonic
stem cell line H1 cultured on micro-carriers to hormone producing
pancreatic cells. Percent positive cells were determined by FACS
shown on the Y-axis for pancreatic hormone cell markers,
Synaptophysin, Glucagon and Insulin. H1 cells were seeded at two
different concentrations 10.times.10.sup.5 (10) or
20.times.10.sup.5 (20) onto Cytodex 3.RTM. (C-3) micro-carriers.
The cells were differentiated in DMEM-High Glucose (HG) during days
four to nine and further differentiated in either HG or DMEM-F12
(F12) media from days 10 through 24.
[0050] FIG. 33: Differentiation of H1 cells on Cytodex 3.RTM.
micro-carriers (GE Healthcare Life Sciences, NJ) to endocrine
cells. H1 cells were differentiated to pancreatic endocrine cells
through pancreatic endoderm (Day 14), pancreatic endocrine cells
(Day 21) to insulin-expressing cells (Day 28). Gene expression
levels of Pdx1, Glucagon, and Insulin were measured (A,B,C
respectively). H1 cells grown and differentiated on Cytodex 3.RTM.
micro-carriers (GE Healthcare Life Sciences, NJ) (C3) were compared
to those grown and differentiated on MATRIGEL (BD Biosciences, CA)
coated 6 well dishes (planar). The gene expression values for cells
grown on Cytodex 3.RTM. micro-carriers (GE Healthcare Life
Sciences, NJ) was performed in triplicate.
[0051] FIG. 34: H9 cells were differentiated on Cytodex 3.RTM.
micro-carriers (GE Healthcare Life Sciences, NJ) to definitive
endoderm (DE). FACS plots of CXCR4 expression. Percent of
definitive endoderm marker CXCR4 positive cells is stated in upper
right corner. A, H9 cells at passage 39 were grown on a MATRIGEL
(BD Biosciences, CA) coated 6 well dishes and differentiated to DE.
B, C Duplicate samples of H9 cells on Cytodex 3.RTM. micro-carriers
(GE Healthcare Life Sciences, NJ) from spinners were placed in a 12
well dish and incubated on a rocking platform.
[0052] FIG. 35: Differentiation of H9 cells on Cytodex 3.RTM.
micro-carriers (GE Healthcare Life Sciences, NJ) to
insulin-expressing cells. H9 cells were differentiated to
pancreatic endocrine cells through pancreatic endoderm (Day 14),
Endocrine cells (Day 22) to Insulin-expressing cells (Day 29). Gene
expression level of Pdx1, Glucagon, and Insulin was measured (A, B,
C respectively). H9 cells grown and differentiated on Cytodex
3.RTM.micro-carriers (GE Healthcare Life Sciences, NJ) (C3) were
compared to those grown and differentiated on MATRIGEL (BD
Biosciences, CA) coated 6 well dishes (planar).
[0053] FIG. 36: Maintenance of pluripotency in human embryonic stem
cells cultured for 5 passages on Cytodex 3.RTM. micro-carriers,
then transferred and cultured on the planar substrates indicated
and cultured in the presence of a Rho kinase inhibitor. Panel A
depicts the expression of the pluripotency markers CD9, SSEA3,
SSEA4, Tra-160, and Tra-181 as detected by flow cytometry. Panel B
depicts the expression of the pluripotency markers Nanog, Pou5F1,
SOX2, and ZFP42 and markers of differentiation: FOXA2, FOXD3,
GATA2, GATA4, and Brachyury as detected by real-time PCR.
[0054] FIG. 37: Formation of definitive endoderm by human embryonic
stem cells cultured for 5 passages on Cytodex 3.RTM.
micro-carriers, then transferred and cultured on the planar
substrates indicated and cultured in the presence of a Rho kinase
inhibitor. Panel A depicts the expression of CXCR4 as detected by
flow cytometry. Panel B depicts the expression of the markers
indicated as detected by real-time PCR.
[0055] FIG. 38: Formation of definitive endoderm by human embryonic
stem cells cultured for 5 passages on Cytodex 3.RTM.
micro-carriers, then transferred and cultured on a PRIMARIA.TM.
planar substrate. Expression of the genes indicated was determined
by flow cytometry.
[0056] FIG. 39: Human embryonic stem cells cultured on planar
substrates maintain pluripotency. mRNA samples from TrypLE.TM.,
Accutase.TM., or Collagenase passaged H1 human ES cells were
collected and assayed for mRNA pluripotency gene expression. Cells
were grown for either one passage for 4 days in culture on MATRIGEL
in MEF conditioned media (A) or one passage on Primaria.TM. in MEF
conditioned media supplemented with Rock Inhibitor (B), or two
passages on Primaria.TM. in MEF conditioned media supplemented with
Rock Inhibitor (C).
[0057] FIG. 40: H1 human embryonic stem cells grown for greater
than 7 passages on PRIMARIA (greater than p45) passaged with
Accutase.TM. or TrypLE.TM. at 1:4, 1:8, or 1:16 split ratios on
PRIMARIA in the presence of Rho Kinase inhibitor Glycyl-H 1152
dihydrochloride were tested for pluripotency (A), and the ability
to differentiate to Definitive Endoderm (B). The control is H1p48
human embryonic stem cells grown on 1:30 MATRIGEL passaged with
collagenase. 10 mA=passaged with 10 minute exposure to
Accutase.TM.. 10 mT=passaged with 10 minute exposure to TrypLE.TM..
1:4, 1:8, or 1:16 indicate the passage ratio. P(X) indicate passage
number since moving from MEF feeders to Primaria.TM. plastic.
[0058] FIG. 41: H1 human embryonic stem cells grown for greater
than 7 passages on PRIMARIA (greater than p45) passaged with
Accutase.TM. or TrypLE.TM. at 1:4 ratio on PRIMARIA in the presence
of Rho Kinase inhibitor Glycyl-H 1152 dihydrochloride were tested
for mRNA expression of pluripotency and differentiation markers.
The control is the starting population of cells at passage 37. 10
min Accutase.TM.=passaged with 10 minute exposure to Accutase.TM..
P(X) indicate passage number since moving from MEF feeders to
PRIMARIA.TM. plastic.
[0059] FIG. 42: H1 human embryonic stem cells grown for greater
than 7 passages on PRIMARIA.TM. (greater than p45) passaged with
Accutase.TM. or TrypLE.TM. at 1:8 ratio on PRIMARIA in the presence
of Rho Kinase inhibitor Glycyl-H 1152 dihydrochloride were tested
for mRNA expression of pluripotency and differentiation markers.
The control is the starting population of cells at passage 37. 10
min Accutase.TM.=passaged with 10 minute exposure to Accutase.TM..
P(X) indicate passage number since moving from MEF feeders to
PRIMARIA.TM. plastic.
[0060] FIG. 43: H1 human embryonic stem cells grown for greater
than 7 passages on PRIMARIA.TM. (greater than p45) passaged with
Accutase.TM. or TrypLE.TM. at 1:16 ratio on PRIMARIA in the
presence of Rho Kinase inhibitor Glycyl-H 1152 dihydrochloride were
tested for mRNA expression of pluripotency and differentiation
markers. The control is the starting population of cells at passage
37. 10 min Accutase.TM.=passaged with 10 minute exposure to
Accutase.TM.. P(X) indicate passage number since moving from MEF
feeders to PRIMARIA.TM. plastic.
[0061] FIG. 44: Images of H1 cells grown on Primaria.TM. planar
substrates (cat. no. 353846, Becton Dickinson, Franklin Lakes,
N.J.) then transferred to micro-carriers 3 days after seeding. A-C
H1 cells were seeded onto Cytodex 3.RTM. (GE Healthcare Life
Sciences, NJ) micro-carriers. D-F Cells were seeded onto
HILLEX.RTM.II micro-carriers (Solohill, MI). A,D H1 cells were
passaged on Primaria.TM. planar substrate (cat. no. 353846, Becton
Dickinson, Franklin Lakes, N.J.) plates with 10 minutes of
TrypLE.TM. Express (Invitrogen, CA) treatment prior to transferring
onto micro-carriers. A,E H1 cells were passaged on Primaria.TM.
planar substrates (cat. no. 353846, Becton Dickinson, Franklin
Lakes, N.J.) plates with 10 minutes of Accutase.TM. treatment prior
to transferring onto micro-carriers. C,F H1 cells at passage 46
were passaged on MATIRGEL (BD Biosciences, CA) coated plates with
collagenase (1 mg/ml) prior to transferring onto
micro-carriers.
[0062] FIG. 45: Pluripotentency of H1 cells grown on Primaria.TM.
planar substrates (cat. no. 353846, Becton Dickinson, Franklin
Lakes, N.J.) then transferred to Cytodex 3.RTM. (GE Healthcare Life
Sciences, NJ) and HILLEX.RTM.II micro-carriers. FACS analysis shows
expression of pluripotent cell-surface proteins. Cells were treated
with Accutase.TM. or TrypLE.TM. Express (Invitrogen, CA) for 3 to
10 minutes during passaging on Primaria.TM. (cat. no. 353846,
Becton Dickinson, Franklin Lakes, N.J.).
[0063] FIG. 46: Differentiation of H1 cells propagated on
Primaria.TM. (cat. no. 353846, Becton Dickinson, Franklin Lakes,
N.J.) then transferred to Cytodex 3.RTM. micro-carriers (GE
Healthcare Life Sciences, NJ). FACS analysis of cell surface
expression of CXCR4, definitive endoderm marker. Cells were treated
with Accutase.TM. or TrypLE.TM. Express (Invitrogen, CA) for 3 to
10 minutes during passaging on Primaria.TM. (cat. no. 353846,
Becton Dickinson, Franklin Lakes, N.J.).
[0064] FIG. 47: FACS analysis of human embryonic stem cells
cultured on planar substrates consisting of mixed cellulose esters
prior to culture on micro-carriers.
[0065] FIG. 48: FACS analysis of the expression of markers
characteristic of the definitive endoderm lineage from human
embryonic stem cells cultured on planar substrates consisting of
mixed cellulose esters prior to culture and differentiation on
micro-carriers.
DETAILED DESCRIPTION
[0066] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the following
subsections that describe or illustrate certain features,
embodiments or applications of the present invention.
DEFINITIONS
[0067] Stem cells are undifferentiated cells defined by their
ability at the single cell level to both self-renew and
differentiate to produce progeny cells, including self-renewing
progenitors, non-renewing progenitors, and terminally
differentiated cells. Stem cells are also characterized by their
ability to differentiate in vitro into functional cells of various
cell lineages from multiple germ layers (endoderm, mesoderm and
ectoderm), as well as to give rise to tissues of multiple germ
layers following transplantation and to contribute substantially to
most, if not all, tissues following injection into blastocysts.
[0068] Stem cells are classified by their developmental potential
as: (1) totipotent, meaning able to give rise to all embryonic and
extraembryonic cell types; (2) pluripotent, meaning able to give
rise to all embryonic cell types; (3) multipotent, meaning able to
give rise to a subset of cell lineages, but all within a particular
tissue, organ, or physiological system (for example, hematopoietic
stem cells (HSC) can produce progeny that include HSC
(self-renewal), blood cell restricted oligopotent progenitors and
all cell types and elements (e.g., platelets) that are normal
components of the blood); (4) oligopotent, meaning able to give
rise to a more restricted subset of cell lineages than multipotent
stem cells; and (5) unipotent, meaning able to give rise to a
single cell lineage (e.g., spermatogenic stem cells).
[0069] Differentiation is the process by which an unspecialized
("uncommitted") or less specialized cell acquires the features of a
specialized cell such as, for example, a nerve cell or a muscle
cell. A differentiated or differentiation-induced cell is one that
has taken on a more specialized ("committed") position within the
lineage of a cell. The term "committed", when applied to the
process of differentiation, refers to a cell that has proceeded in
the differentiation pathway to a point where, under normal
circumstances, it will continue to differentiate into a specific
cell type or subset of cell types, and cannot, under normal
circumstances, differentiate into a different cell type or revert
to a less differentiated cell type. De-differentiation refers to
the process by which a cell reverts to a less specialized (or
committed) position within the lineage of a cell. As used herein,
the lineage of a cell defines the heredity of the cell, i.e., which
cells it came from and what cells it can give rise to. The lineage
of a cell places the cell within a hereditary scheme of development
and differentiation. A lineage-specific marker refers to a
characteristic specifically associated with the phenotype of cells
of a lineage of interest and can be used to assess the
differentiation of an uncommitted cell to the lineage of
interest.
[0070] Various terms are used to describe cells in culture.
"Maintenance" refers generally to cells placed in a growth medium
under conditions that facilitate cell growth and/or division that
may or may not result in a larger population of the cells.
"Passaging" refers to the process of removing the cells from one
culture vessel and placing them in a second culture vessel under
conditions that facilitate cell growth and/or division.
[0071] A specific population of cells, or a cell line, is sometimes
referred to or characterized by the number of times it has been
passaged. For example, a cultured cell population that has been
passaged ten times may be referred to as a P10 culture. The primary
culture, i.e., the first culture following the isolation of cells
from tissue, is designated P0. Following the first subculture, the
cells are described as a secondary culture (P1 or passage 1). After
the second subculture, the cells become a tertiary culture (P2 or
passage 2), and so on. It will be understood by those of skill in
the art that there may be many population doublings during the
period of passaging; therefore the number of population doublings
of a culture is greater than the passage number. The expansion of
cells (i.e., the number of population doublings) during the period
between passaging depends on many factors, including but not
limited to the seeding density, substrate, medium, growth
conditions, and time between passaging.
[0072] ".beta.-cell lineage" refer to cells with positive gene
expression for the transcription factor PDX-1 and at least one of
the following transcription factors: NGN-3, NRx2.2, NRx6.1, NeuroD,
Isl-1, HNF-3 beta, MAFA, Pax4, or Pax6. Cells expressing markers
characteristic of the .beta. cell lineage include .beta. cells.
[0073] "Cells expressing markers characteristic of the definitive
endoderm lineage" as used herein refer to cells expressing at least
one of the following markers: SOX-17, GATA-4, HNF-3 beta, GSC,
Cer1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48,
eomesodermin (EOMES), DKK4, FGF17, GATA-6, CXCR4, C-Kit, CD99, or
OTX2. Cells expressing markers characteristic of the definitive
endoderm lineage include primitive streak precursor cells,
primitive streak cells, mesendoderm cells and definitive endoderm
cells.
[0074] "Cells expressing markers characteristic of the pancreatic
endoderm lineage" as used herein refer to cells expressing at least
one of the following markers: PDX-1, HNF-1beta, PTF-1 alpha, HNF-6,
or HB9. Cells expressing markers characteristic of the pancreatic
endoderm lineage include pancreatic endoderm cells.
[0075] "Cells expressing markers characteristic of the pancreatic
endocrine lineage" as used herein refer to cells expressing at
least one of the following markers: NGN-3, NeuroD, Islet-1, PDX-1,
NKX6.1, Pax-4, Ngn-3, or PTF-1 alpha. Cells expressing markers
characteristic of the pancreatic endocrine lineage include
pancreatic endocrine cells, pancreatic hormone expressing cells,
and pancreatic hormone secreting cells, and cells of the
.beta.-cell lineage.
[0076] "Definitive endoderm" as used herein refers to cells which
bear the characteristics of cells arising from the epiblast during
gastrulation and which form the gastrointestinal tract and its
derivatives. Definitive endoderm cells express the following
markers: CXCR4, HNF-3 beta, GATA-4, SOX-17, Cerberus, OTX2,
goosecoid, c-Kit, CD99, and Mixl1.
[0077] "Extraembryonic endoderm" as used herein refers to a
population of cells expressing at least one of the following
markers: SOX-7, AFP, or SPARC.
[0078] "Markers" as used herein, are nucleic acid or polypeptide
molecules that are differentially expressed in a cell of interest.
In this context, differential expression means an increased level
for a positive marker and a decreased level for a negative marker.
The detectable level of the marker nucleic acid or polypeptide is
sufficiently higher or lower in the cells of interest compared to
other cells, such that the cell of interest can be identified and
distinguished from other cells using any of a variety of methods
known in the art.
[0079] "Mesendoderm cell" as used herein refers to a cell
expressing at least one of the following markers: CD48,
eomesodermin (EOMES), SOX-17, DKK4, HNF-3 beta, GSC, FGF17, or
GATA-6.
[0080] "Pancreatic endocrine cell" or "pancreatic hormone
expressing cell" as used herein refers to a cell capable of
expressing at least one of the following hormones: insulin,
glucagon, somatostatin, and pancreatic polypeptide.
[0081] "Pancreatic hormone secreting cell" as used herein refers to
a cell capable of secreting at least one of the following hormones:
insulin, glucagon, somatostatin, and pancreatic polypeptide.
[0082] "Pre-primitive streak cell" as used herein refers to a cell
expressing at least one of the following markers: Nodal, or
FGF8.
[0083] "Primitive streak cell" as used herein refers to a cell
expressing at least one of the following markers: Brachyury,
Mix-like homeobox protein, or FGF4.
Micro-Carriers
[0084] "Micro-carriers" refers to particles, beads, or pellets
useful for attachment and growth of anchorage dependent cells in
culture. The micro-carriers have the following properties: (a) They
are small enough to allow them to be used in suspension cultures
(with a stirring rate that does not cause significant shear damage
to the micro-carriers or the cells); (b) They are solid, or have a
solid core with a porous coating on the surface; and (c) Their
surfaces (exterior and interior surface in case of porous carriers)
may be positively or negatively charged. In one aspect, the
micro-carriers have an overall particle diameter between about 150
and 350 .mu.m, and have a positive charge density of between about
0.8 and 2.0 meq/g. Useful micro-carriers include, without
limitation, Cytodex 1.RTM., Cytodex 2.RTM., or Cytodex 3.RTM. (GE
Healthcare Life Sciences, NJ).
[0085] In another aspect, the micro-carrier is a solid carrier.
Solid carriers are particularly suitable for adhesion cells, e.g.,
anchorage-dependent cells. The carrier particle can also be a
porous micro-carrier.
[0086] "Porous micro-carriers" refers to particles useful for
attachment and growth of anchorage-dependent cells in culture. The
porous micro-carriers have the following properties: (a) they are
small enough to allow them to be used in suspension cultures (with
a stirring rate that does not cause significant shear damage to the
micro-carriers or the cells); (b) they have pores and interior
spaces of sufficient size to allow cells to migrate into the
interior spaces of the particle and (c) their surfaces (exterior
and interior) may be positively or negatively charged. In one
series of embodiments, the carriers (a) have an overall particle
diameter between about 150 and 350 .mu.m; (b) have pores having an
average pore opening diameter of between about 15 and about 40
.mu.m; and (c) have a positive charge density of between about 0.8
and 2.0 meq/g. In some embodiments, the positive charge is provided
by DEAE (N,N,-diethylaminoethyl) groups. Useful porous
micro-carriers include, without limitation, Cytopore 1.RTM. and
Cytopore 2.RTM. (GE Healthcare Life Sciences, Piscataway N.J.).
Micro-carriers may be any shape, but are typically roughly
spherical in shape, and can be either macro- or micro-porous, or
solid.
[0087] Both porous and solid types of micro-particulate carriers
are commercially available from suppliers. Examples of commercially
available micro-carriers include Cytodex 10 and Cytodex 3.RTM. (GE
Healthcare Life Sciences, NJ), which are both dextran-based
micro-carriers from GE Healthcare Life Sciences. Porous
micro-carriers on the market include Cytoline as well as Cytopore
products also from GE Healthcare Life Sciences. Biosilon (NUNC) and
Cultispher (Percell Biolytica) are also commercially available. In
a further aspect, the micro-carriers can be comprised of, or coated
with polycarbonate or mixed cellulose esters.
[0088] Micro-carriers suitable for use in the present invention can
be comprised of natural or synthetically-derived materials.
Examples include collagen-based micro-carriers, dextran-based
micro-carriers, or cellulose-based micro-carriers, as well as
glass, ceramics, polymers, or metals. The micro-carrier can be
protein-free or protein-coated, for example, with collagen. In a
further aspect the micro-carrier can be comprised of, or coated
with, compounds that enhance binding of the cell to the
micro-carrier and enhance release of the cell from the
micro-carrier including, but not limited to,
poly(monostearoylglyceride co-succinic acid),
poly-D,L-lactide-co-glycolide, sodium hyaluronate, collagen,
fibronectin, laminin, elastin, lysine, n-isopropyl acrylamide,
vitronectin.
Micro-Carriers for Cell Culture
[0089] Micro-carrier culture is a technique, which makes possible
the practical high yield culture of anchorage-dependent, cells, for
example, human embryonic stem cells. Micro-carriers have been
specifically developed for the culture of cells, such as human
embryonic stem cells, in culture volumes ranging from a few
milliliters to greater than one thousand liters. The micro-carrier
is biologically inert and provides a strong but non-rigid substrate
for stirred micro-carrier cultures. The micro-carriers may be
transparent, allowing microscopic examination of the attached
cells. Cytodex 3.RTM. (GE Healthcare Life Sciences, NJ) consists of
a thin layer of denatured collagen chemically coupled to a matrix
of crosslinked dextran. The denatured collagen layer on Cytodex
3.RTM. (GE Healthcare Life Sciences, NJ) is susceptible to
digestion by a variety of proteases, including trypsin and
collagenase, and provides the ability to remove cells from the
micro-carriers while maintaining maximum cell viability, function,
and integrity.
[0090] Protein free micro-carriers can be used to culture human
embryonic stem cells. For example, micro-carriers for use in
manufacturing and laboratory or research use sold under the
tradename HILLEX.RTM. (SoloHill Engineering, Inc., MI.) are
modified polystyrene beads with cationic trimethyl ammonium
attached to the surface to provide a positively charged surface to
the micro-carrier. The bead diameter ranges from about 90 to about
200 microns in diameter.
[0091] Micro-carrier-based methods of cell culture provided many
advantages including ease of downstream processing in many
applications. Micro-carriers are typically roughly spherical in
shape, and can be either porous or solid. The use of micro-carriers
for cell attachment facilitates the use of stirred tank and related
reactors for growth of anchorage-dependent cells. The cells attach
to the readily suspended micro-carriers. The requirement for
suspendability limits the physical parameters of the
micro-carriers. Thus, micro-carriers commonly have a mean diameter
in the range of 50-2000 microns. In some applications solid-type
micro-carriers range from about 100 to about 250 microns whereas
porous-type micro-carriers range from about 250 to about 2500
microns. These size ranges allow for selection of micro-carriers,
which are large enough to accommodate many anchorage-dependent
cells, while small enough to form suspensions with properties
suitable for use in stirred reactors.
[0092] Among the factors considered in using micro carriers and the
like are: attachment efficiency, immunogenicity, biocompatibility,
ability to biodegrade, time to reach confluence, the growth
parameters of attached cells including maximum attainable density
per unit surface area, detachment techniques where required, and
the efficiency of the detachment, scalability of the culture
conditions as well as homogeneity of the culture under scaled-up
conditions, the ability to successfully scale-up detachment
procedures, and whether the micro-carriers will be used for
implantation. These considerations can be influenced by the surface
properties of the micro-carrier, as well as by the porosity,
diameter, density, and handling properties of the
micro-carrier.
[0093] For example, the density of the micro-carriers is a
consideration. Excessive density may cause the micro-carriers to
settle out of the suspension, or tend to remain completely towards
the bottom of the culture vessel, and thus may result in poor bulk
mixing of the cells, culture medium and gaseous phases in the
reactor. On the other hand, a density that is too low may result in
excessive floating of the micro-carrier. A density of 1.02 to 1.15
g/cm.sup.3 is typical of many micro-carriers.
[0094] The small diameter of micro-carriers and the volume of
particles that can be added to a reactor allows the micro-carriers
to contribute substantial surface area in vast excess to that found
in roller bottles or other methods of growing anchorage-dependent
cells, e.g. on plates. Porous micro-carriers provide even greater
surface area per unit volume or weight. These porous micro-carriers
possess large cavities that are available for the growth of
anchorage-dependent cells. These cavities increase the surface area
greatly, and may protect cells from detrimental mechanical effects,
such as shear stress, for example from mixing or from gas
sparging.
[0095] The micro-carrier surface may be textured to enhance cell
attachment and proliferation. The micro-carrier surface texture be
achieved by techniques including, but not limited to, molding,
casting, leeching and etching. The resolution of the features of
the textured surface may be on the nanoscale. The textured surface
may be used to induce a specific cell alignment on the
micro-carrier surface. The surface of the pores within the porous
micro-carriers may also be textured to enhance cell attachment and
proliferation. Pore surface texture be achieved by techniques such
as but not limited to molding, casting, leeching and etching.
[0096] The micro-carrier surface may be plasma-coated to impart a
specific charge to micro-carrier surfaces. These charges may
enhance cell attachment and proliferation.
[0097] In other embodiments, the micro-carriers are composed of, or
coated with, thermoresponsive polymers such as
poly-N-isopropylacrylamide, or have electromechanical
properties.
[0098] Both porous and solid types of microparticulate carriers are
commercially available from suppliers. Examples of commercially
available solid micro-carriers include Cytodex 1.RTM. and Cytodex
3.RTM. (GE Healthcare Life Sciences, NJ), which are both
dextran-based micro-carriers from GE Healthcare Life Sciences.
Porous micro-carriers on the market include Cytoline as well as
Cytopore products also from GE Healthcare Life Sciences. Biosilon
(NUNC) and Cultispher (Percell Biolytica) are also commercially
available.
[0099] The micro-carriers may also contain a bioactive agent. The
micro-carrier may also contain a bioactive agent that may regulate
the growth or function of cells or the tissue milieu these factors
may include but are not limited to fibroblast growth factors,
erythropoietin, vascular endothelial cell growth factors, platelet
derived growth factors, bone morphogenic proteins, transforming
growth factors, tumor necrosis factors, epidermal growth factors,
insulin-like growth factors. Complete factors, mimetics or active
fragments thereof may be used.
[0100] The micro-carriers may be inoculated with a second cell type
and co-cultured with the pluripotent stem cells. In one embodiment
the two (or more) cell types may be adherent to an individual
micro-carrier in equal or un-equal proportions. The two or more
cell types can be inoculated onto the micro-carrier at the same
time point or they may be inoculated at different times. The
micro-carriers can be treated in such a manner to preferentially
adhere specific cell types onto specific regions of the
micro-carrier. In a further embodiment, the micro-carrier with
adherent single or multiple cell types can be co-cultured in a
culture vessel with a second cell type cultured in suspension.
[0101] Second cell types may include, for example, epithelial cells
(e.g., cells of oral mucosa, gastrointestinal tract, nasal
epithelium, respiratory tract epithelium, vaginal epithelium,
corneal epithelium), bone marrow cells, adipocytes, stem cells,
keratinocytes, melanocytes, dermal fibroblasts, keratinocytes,
vascular endothelial cells (e.g., aortic endothelial cells,
coronary artery endothelial cells, pulmonary artery endothelial
cells, iliac artery endothelial cells, microvascular endothelial
cells, umbilical artery endothelial cells, umbilical vein
endothelial cells, and endothelial progenitors (e.g., CD34+,
CD34+/CD117+ cells)), myoblasts, myocytes, hepatocytes, smooth
muscle cells, striated muscle cells, stromal cells, and other soft
tissue cells or progenitor cells, chondrocytes, osteoblasts, islet
cells, nerve cells including but not limited to neurons,
astrocytes, Schwann cells, enteric glial cells,
oligodendrocytes.
Pluripotent Stem Cells
Characterization of Pluripotent Stem Cells
[0102] Pluripotent stem cells may express one or more of the
stage-specific embryonic antigens (SSEA) 3 and 4, and markers
detectable using antibodies designated Tra-1-60 and Tra-1-81
(Thomson et al., Science 282:1145, 1998). Differentiation of
pluripotent stem cells in vitro results in the loss of SSEA-4,
Tra-1-60, and Tra-1-81 expression (if present) and increased
expression of SSEA-1. Undifferentiated pluripotent stem cells
typically have alkaline phosphatase activity, which can be detected
by fixing the cells with 4% paraformaldehyde and then developing
with Vector Red as a substrate, as described by the manufacturer
(Vector Laboratories, Burlingame Calif.) Undifferentiated
pluripotent stem cells also typically express OCT4 and TERT, as
detected by RT-PCR.
[0103] Another desirable phenotype of propagated pluripotent stem
cells is a potential to differentiate into cells of all three
germinal layers: endoderm, mesoderm, and ectoderm tissues.
Pluripotency of stem cells can be confirmed, for example, by
injecting cells into severe combined immunodeficient (SCID) mice,
fixing the teratomas that form using 4% paraformaldehyde, and then
examining them histologically for evidence of cell types from the
three germ layers. Alternatively, pluripotency may be determined by
the creation of embryoid bodies and assessing the embryoid bodies
for the presence of markers associated with the three germinal
layers.
[0104] Propagated pluripotent stem cell lines may be karyotyped
using a standard G-banding technique and compared to published
karyotypes of the corresponding primate species. It is desirable to
obtain cells that have a "normal karyotype," which means that the
cells are euploid, wherein all human chromosomes are present and
not noticeably altered.
Sources of Pluripotent Stem Cells
[0105] The types of pluripotent stem cells that may be used include
established lines of pluripotent cells derived from tissue formed
after gestation, including pre-embryonic tissue (such as, for
example, a blastocyst), embryonic tissue, or fetal tissue taken any
time during gestation, typically but not necessarily before
approximately 10-12 weeks gestation. Non-limiting examples are
established lines of human embryonic stem cells or human embryonic
germ cells, such as, for example the human embryonic stem cell
lines H1, H7, and H9 (WiCell). Also contemplated is use of the
compositions of this disclosure during the initial establishment or
stabilization of such cells, in which case the source cells would
be primary pluripotent cells taken directly from the source
tissues. Also suitable are cells taken from a pluripotent stem cell
population already cultured in the absence of feeder cells. Also
suitable are mutant human embryonic stem cell lines, such as, for
example, BG01v (BresaGen, Athens, Ga.). Also suitable are
pluripotent stem cells derived from non-pluripotent cells, such as,
for example, an adult somatic cells.
Attaching Pluripotent Stem Cells to the Micro-Carriers Suitable for
Use in the Present Invention
[0106] Pluripotent stem cells may be cultured on a planar substrate
by any method in the art, prior to attaching to micro-carriers. For
example, pluripotent stem cells may be cultured on planar
substrates, treated with an extracellular matrix protein (e.g.
MATRIGEL). Alternatively, pluripotent stem cells may be cultured on
planar substrates seeded with a feeder cell layer.
[0107] In one embodiment, the pluripotent stem cells are embryonic
stem cells. In an alternate embodiment, the embryonic stem cells
are human.
[0108] In one aspect of the present invention, pluripotent stem
cells are released from a planar substrate by treating the
pluripotent stem cells with a protease that will release the cells
from the planar substrate. The protease may be, for example,
collagenase, TrypLE.TM. Express, Accutase.TM., trypsin, and the
like.
[0109] In one embodiment, the pluripotent stem cells are released
from the micro-carrier substrate by treating the cells with
Accutase.TM. for about five to about ten minutes.
[0110] In one embodiment, the pluripotent stem cells are released
from the micro-carrier substrate by treating the cells with 0.05%
trypsin/EDTA for about ten to about twenty minutes.
[0111] In one embodiment, the pluripotent stem cells are released
from the micro-carrier substrate by treating the cells with
TrypLE.TM. Express for about five to about twenty minutes.
[0112] In one embodiment, the pluripotent stem cells are released
from the micro-carrier substrate by treating the cells with 10
mg/ml Collagenase for about five to about ten minutes.
[0113] The released pluripotent cells are added to medium
containing micro-carriers at a specific density. In one embodiment,
the pluripotent stem cells were seeded at about 4,000 to about
30,000 cells per cm.sup.2 of micro-carriers.
[0114] The released pluripotent cells are added to medium
containing micro-carriers. In one embodiment, the attachment of the
pluripotent stem cells is enhanced by treating the pluripotent stem
cells with a Rho kinase inhibitor. The Rho kinase inhibitor may be
Y27632 (Sigma-Aldrich, MO). Alternatively, the Rho kinase inhibitor
is Glycyl-H 1152 dihydrochloride.
[0115] In one embodiment, the pluripotent stem cells are treated
with Y27632 at a concentration from about 1 .mu.M to about 10
.mu.M. In one embodiment, the pluripotent stem cells are treated
with Y27632 at a concentration of about 10 .mu.M.
[0116] In one embodiment, the pluripotent stem cells are treated
with Glycyl-H 1152 dihydrochloride at a concentration from about
0.25 .mu.M to about 5 .mu.M. In one embodiment, the pluripotent
stem cells are treated with Glycyl-H 1152 dihydrochloride at a
concentration of about 2.5 .mu.M.
[0117] The medium containing the micro-carriers may be agitated.
Agitation as used in the present invention may be the movement of
the culture medium. Such agitation may be achieved manually, or,
alternatively, by use of apparatus, such as, for example, a rocking
platform, a spinner flask, and the like. In one embodiment, the
medium containing the micro-carriers is agitated by the use of
manual movement. The dish containing the micro-carriers and cells
is moved back and forth for less than 30 seconds.
[0118] The medium containing the micro-carriers may be agitated. In
one embodiment, the medium containing the micro-carriers is
agitated by the use of a spinner flask. The spinner flask (Corning,
Lowell, Mass.) is placed on a stir plate at 30-70 RPM depending on
bead type.
[0119] In an alternate embodiment, the medium containing the
micro-carriers is agitated by the use of a rocking platform
(Vari-mix, Barnstead, Dubuque, Iowa). The rocking platform speed is
about one rotation in 2 seconds.
Differentiating Pluripotent Stem Cells on Micro-Carriers
[0120] In one embodiment, the pluripotent stem cells may be
differentiated into cells expressing markers characteristic of the
definitive endoderm lineage on micro-carriers. Alternatively, the
pluripotent stem cells may be differentiated into cells expressing
markers characteristic of the pancreatic endoderm lineage on
micro-carriers. Alternatively, the pluripotent stem cells may be
differentiated into cells expressing markers characteristic of the
pancreatic endocrine lineage on micro-carriers.
[0121] In an alternate embodiment, the pluripotent stem cells may
be propagated on micro-carriers, then differentiated into cells
expressing markers characteristic of the definitive endoderm
lineage on planar surfaces. Alternatively, the pluripotent stem
cells may be propagated on micro-carriers, then differentiated into
cells expressing markers characteristic of the pancreatic endoderm
lineage on planar surfaces. Alternatively, the pluripotent stem
cells may be propagated on micro-carriers, then differentiated into
cells expressing markers characteristic of the pancreatic endocrine
lineage on planar surfaces.
[0122] Pluripotent stem cells treated in accordance with the
methods of the present invention may be differentiated into a
variety of other cell types by any suitable method in the art. For
example, pluripotent stem cells treated in accordance with the
methods of the present invention may be differentiated into neural
cells, cardiac cells, hepatocytes, and the like.
[0123] For example, pluripotent stem cells treated in accordance
with the methods of the present invention may be differentiated
into neural progenitors and cardiomyocytes according to the methods
disclosed in WO2007030870.
[0124] In another example, pluripotent stem cells treated in
accordance with the methods of the present invention may be
differentiated into hepatocytes according to the methods disclosed
in U.S. Pat. No. 6,458,589.
Formation of Cells Expressing Markers Characteristic of the
Definitive Endoderm Lineage
[0125] Pluripotent stem cells may be differentiated into cells
expressing markers characteristic of the definitive endoderm
lineage by any method in the art.
[0126] For example, pluripotent stem cells may be differentiated
into cells expressing markers characteristic of the definitive
endoderm lineage according to the methods disclosed in D'Amour et
al, Nature Biotechnology 23, 1534-1541 (2005).
[0127] For example, pluripotent stem cells may be differentiated
into cells expressing markers characteristic of the definitive
endoderm lineage according to the methods disclosed in Shinozaki et
al, Development 131, 1651-1662 (2004).
[0128] For example, pluripotent stem cells may be differentiated
into cells expressing markers characteristic of the definitive
endoderm lineage according to the methods disclosed in McLean et
al, Stem Cells 25, 29-38 (2007).
[0129] For example, pluripotent stem cells may be differentiated
into cells expressing markers characteristic of the definitive
endoderm lineage according to the methods disclosed in D'Amour et
al, Nature Biotechnology 24, 1392-1401 (2006).
[0130] Markers characteristic of the definitive endoderm lineage
are selected from the group consisting of SOX17, GATA4, HNF-3 beta,
GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4,
CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99,
and OTX2. Suitable for use in the present invention is a cell that
expresses at least one of the markers characteristic of the
definitive endoderm lineage. In one aspect of the present
invention, a cell expressing markers characteristic of the
definitive endoderm lineage is a primitive streak precursor cell.
In an alternate aspect, a cell expressing markers characteristic of
the definitive endoderm lineage is a mesendoderm cell. In an
alternate aspect, a cell expressing markers characteristic of the
definitive endoderm lineage is a definitive endoderm cell.
[0131] In another example, pluripotent stem cells treated according
to the methods of the present invention may be differentiated into
cells expressing markers characteristic of the definitive endoderm
lineage by culturing the pluripotent stem cells in medium
containing activin A in the absence of serum, then culturing the
cells with activin A and serum, and then culturing the cells with
activin A and serum of a different concentration. An example of
this method is disclosed in Nature Biotechnology 23, 1534-1541
(2005).
[0132] In another example, pluripotent stem cells treated according
to the methods of the present invention may be differentiated into
cells expressing markers characteristic of the definitive endoderm
lineage by culturing the pluripotent stem cells in medium
containing activin A in the absence of serum, then culturing the
cells with activin A with serum of another concentration. An
example of this method is disclosed in D' Amour et al, Nature
Biotechnology, 2005.
[0133] In another example, pluripotent stem cells treated according
to the methods of the present invention may be differentiated into
cells expressing markers characteristic of the definitive endoderm
lineage by culturing the pluripotent stem cells in medium
containing activin A and a Wnt ligand in the absence of serum, then
removing the Wnt ligand and culturing the cells with activin A with
serum. An example of this method is disclosed in Nature
Biotechnology 24, 1392-1401 (2006).
[0134] In another example, pluripotent stem cells treated according
to the methods of the present invention may be differentiated into
cells expressing markers characteristic of the definitive endoderm
lineage according to the methods disclosed in U.S. patent
application Ser. No. 11/736,908, assigned to LifeScan, Inc.
[0135] In another example, pluripotent stem cells treated according
to the methods of the present invention may be differentiated into
cells expressing markers characteristic of the definitive endoderm
lineage according to the methods disclosed in U.S. patent
application Ser. No. 11/779,311, assigned to LifeScan, Inc.
Formation of Cells Expressing Markers Characteristic of the
Pancreatic Endoderm Lineage
[0136] Pluripotent stem cells may be differentiated into cells
expressing markers characteristic of the pancreatic endoderm
lineage by any method in the art.
[0137] For example, pluripotent stem cells may be differentiated
into cells expressing markers characteristic of the pancreatic
endoderm lineage according to the methods disclosed in D'Amour et
al, Nature Biotechnology 24, 1392-1401 (2006).
[0138] For example, cells expressing markers characteristic of the
definitive endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endoderm
lineage, by treating the cells expressing markers characteristic of
the definitive endoderm lineage with a fibroblast growth factor and
the hedgehog signaling pathway inhibitor KAAD-cyclopamine, then
removing the medium containing the fibroblast growth factor and
KAAD-cyclopamine and subsequently culturing the cells in medium
containing retinoic acid, a fibroblast growth factor and
KAAD-cyclopamine. An example of this method is disclosed in Nature
Biotechnology 24, 1392-1401 (2006).
[0139] For example, cells expressing markers characteristic of the
definitive endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endoderm
lineage, by treating the cells expressing markers characteristic of
the definitive endoderm lineage with retinoic acid one fibroblast
growth factor for a period of time, according to the methods
disclosed in U.S. patent application Ser. No. 11/736,908, assigned
to LifeScan, Inc.
[0140] For example, cells expressing markers characteristic of the
definitive endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endoderm
lineage, by treating the cells expressing markers characteristic of
the definitive endoderm lineage with retinoic acid (Sigma-Aldrich,
MO) and exendin 4, then removing the medium containing DAPT
(Sigma-Aldrich, MO) and exendin 4 and subsequently culturing the
cells in medium containing exendin 1, IGF-1 and HGF. An example of
this method is disclosed in Nature Biotechnology 24, 1392-1401
(2006).
[0141] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by culturing the cells expressing markers characteristic
of the pancreatic endoderm lineage in medium containing exendin 4,
then removing the medium containing exendin 4 and subsequently
culturing the cells in medium containing exendin 1, IGF-1 and HGF.
An example of this method is disclosed in D' Amour et al, Nature
Biotechnology, 2006.
[0142] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by culturing the cells expressing markers characteristic
of the pancreatic endoderm lineage in medium containing DAPT
(Sigma-Aldrich, MO) and exendin 4. An example of this method is
disclosed in D' Amour et al, Nature Biotechnology, 2006.
[0143] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by culturing the cells expressing markers characteristic
of the pancreatic endoderm lineage in medium containing exendin 4.
An example of this method is disclosed in D' Amour et al, Nature
Biotechnology, 2006.
[0144] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by treating the cells expressing markers characteristic of
the pancreatic endoderm lineage with a factor that inhibits the
Notch signaling pathway, according to the methods disclosed in U.S.
patent application Ser. No. 11/736,908, assigned to LifeScan,
Inc.
[0145] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by treating the cells expressing markers characteristic of
the pancreatic endoderm lineage with a factor that inhibits the
Notch signaling pathway, according to the methods disclosed in U.S.
patent application Ser. No. 11/779,311, assigned to LifeScan,
Inc.
[0146] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by treating the cells expressing markers characteristic of
the pancreatic endoderm lineage with a factor that inhibits the
Notch signaling pathway, according to the methods disclosed in U.S.
patent application Ser. No. 11/736,908, assigned to LifeScan,
Inc.
[0147] For example, cells expressing markers characteristic of the
pancreatic endoderm lineage obtained according to the methods of
the present invention are further differentiated into cells
expressing markers characteristic of the pancreatic endocrine
lineage, by treating the cells expressing markers characteristic of
the pancreatic endoderm lineage with a factor that inhibits the
Notch signaling pathway, according to the methods disclosed in U.S.
patent application Ser. No. 11/779,311, assigned to LifeScan,
Inc.
[0148] Markers characteristic of the pancreatic endocrine lineage
are selected from the group consisting of NGN3, NEUROD, ISL1, PDX1,
NKX6.1, PAX4, NGN3, and PTF-1 alpha. In one embodiment, a
pancreatic endocrine cell is capable of expressing at least one of
the following hormones: insulin, glucagon, somatostatin, and
pancreatic polypeptide. Suitable for use in the present invention
is a cell that expresses at least one of the markers characteristic
of the pancreatic endocrine lineage. In one aspect of the present
invention, a cell expressing markers characteristic of the
pancreatic endocrine lineage is a pancreatic endocrine cell. The
pancreatic endocrine cell may be a pancreatic hormone-expressing
cell. Alternatively, the pancreatic endocrine cell may be a
pancreatic hormone-secreting cell.
[0149] In one aspect of the present invention, the pancreatic
endocrine cell is a cell expressing markers characteristic of the
.beta. cell lineage. A cell expressing markers characteristic of
the .beta. cell lineage expresses PDX1 and at least one of the
following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD,
ISL1, HNF-3 beta, MAFA, PAX4, or PAX6. In one aspect of the present
invention, a cell expressing markers characteristic of the .beta.
cell lineage is a .beta. cell.
[0150] The present invention is further illustrated, but not
limited by, the following examples.
EXAMPLES
Example 1
Attachment and Proliferation of Human Embryonic Stem Cells on
Micro-Carriers
[0151] To determine if human embryonic stem cells can attach and
proliferate on micro-carriers, H9 cells passage 52 were released
from MATRIGEL.TM. (BD Biosciences, CA) coated plates with
TrypLE.TM. Express. They were then incubated with micro-carriers
and MEF-CM. Suspensions of ProNectinF (PN), Plastic (P),
PlasticPlus (PP), HILLEX.RTM.II (H), collagen (Col) and FACT III
(SoloHill, MI) micro-carriers were prepared according to
manufacturer's instructions. After 2 days at 37.degree. C., Table 1
describes the attachment and growth of the H9 cells on the
micro-carriers based on daily images. Few cells attached and/or
proliferated on most micro-carriers tested. H9 cells did attach and
proliferate on HILLEX.RTM.II micro-carriers (Solohill, MI) but
images showed fewer cell-bead aggregates after 2 days in static
culture (FIG. 1B).
[0152] To improve the attachment and proliferation of human
embryonic stem cells on micro-carriers, a small molecule inhibitor
of Rho-associated coiled coil forming protein serine/threonine
kinase, Rho kinase inhibitor was added to the media. Specifically,
Y27632, Y, (Sigma-Aldrich, MO) was used. MEF-CM plus 10 .mu.M
Y27632 (Sigma-Aldrich, MO) was changed daily. In the presence of 10
.mu.M Y27632 (Sigma-Aldrich, MO) the H9 cells attached and formed
aggregates with all micro-carriers tested (Table 2). By analysis of
images, human embryonic stem cells grown on HILLEX.RTM.II
micro-carriers (Solohill, MI) appeared to attach and proliferate
better than human embryonic stem cells on other micro-carriers
tested. Additionally, H9 cells attached better to HILLEX.RTM.II
(Solohill, MI) in the presence of the Rho kinase inhibitor (FIG. 1A
compared to 1B).
[0153] Expansion of human embryonic stem cells for a cell therapy
application is necessary to meet product demand. Currently the best
techniques for expansion include spinner flasks and bioreactors.
Both of these techniques require physical movement of the
micro-carriers in suspension. To determine the effect of motion on
the growth of the human embryonic stem cells on micro-carriers, 6
or 12 well dishes were placed on a rocking platform in a 37.degree.
C. incubator. After growth for 3 days, the cell aggregates began to
release from some of the micro-carriers. FIG. 2 A, B, D illustrates
that the cell aggregates disassociated from the Plastic Plus,
Plastic, or Pronectin micro-carriers. In contrast, the cells
remained attached to the HILLEX.RTM. II micro-carriers (Solohill,
MI) and proliferated FIG. 2C. Example 4 describes the dissociation
method used prior to cell counting in a Guava PCA-96 with Viacount
Flex (Guava Technologies, Hayward, Calif.). Measuring the growth
rate of the cells on micro-carriers reveals a dip in cell number at
day 3 compared to the starting number at seeding. This is likely
due to poor initial attachment of the cells to the micro-carriers
followed by an expansion afterwards until the experiment was
terminated at day 5. H9 cells on HILLEX.RTM.II micro-carriers
(Solohill, MI) have the highest proliferation rate compared to the
other bead types, likely due to better attachment of the cells to
the HILLEX.RTM. II micro-carriers (Solohill, MI) (FIGS. 2, 3). This
demonstrates that the HILLEX.RTM. II micro-carriers (Solohill, MI)
can support growth of H9 cells in suspension. This was further
validated after repeat passaging, see Example 5.
[0154] The H1 human embryonic cell line was also tested for growth
on micro-carriers for large-scale expansion. Because a Rho kinase
inhibitor, Y27632 (Sigma-Aldrich, MO), was necessary for attachment
of the H9 cell line, it was also assumed to be necessary for H1
cells. Cytodex 1.RTM., Cytodex 3.RTM. (GE Healthcare Life Sciences,
NJ), HILLEX.RTM.II, Plastic, ProNectinF, Plastic Plus
micro-carriers (SoloHill Ann Arbor, Mich.) were prepared according
to the manufacturer's instructions. The H1 human embryonic stem
cells at passage 47 were seeded at about 13,333 cells/cm.sup.2 of
micro-carriers in MEF-CM plus 10 .mu.M Y27632 (Sigma-Aldrich, MO).
Cells and micro-carriers were placed in a 12 well non-tissue
culture treated dish at 15 cm.sup.2 per 12 well on a rocking
platform at 37.degree. C. to allow movement of the micro-carriers
and medium. After 3, 5 and 7 days, one well was imaged, harvested,
and counted. The ability of cells to attach depended on the bead
type. Similar results were observed with the H1 line as with the H9
line. Specifically, cells seeded onto Plastic, Plastic Plus or
ProNectinF micro-carriers did not attach and/or proliferate well
(FIG. 4). Cells seeded onto HILLEX.RTM.II (Solohill, MI), Cytodex
1.RTM., or Cytodex 3.RTM. (GE Healthcare Life Sciences, NJ)
micro-carriers attached and proliferated well (FIG. 5). Cells were
detached according to Example 4 and counted for yield. Cells grown
on Cytodex 3.RTM. micro-carriers (GE Healthcare Life Sciences, NJ)
exhibited the highest cell number after 7 days in culture (FIG.
6).
Example 2
Optimal Concentrations of Y27632 and Other Rho Kinase Inhibitors
for Cell Attachment and Growth
[0155] To determine the concentration of Rho kinase inhibitor that
best supports attachment and growth of the human embryonic stem
cells on micro-carriers, the following experiments were
conducted.
[0156] A starting aliquot of 13,333 cells/cm.sup.2H9 cells at
passage 44 was seeded onto 15 cm.sup.2 of micro-carriers in a
single well of a 12 well non-tissue culture treated plate. The
cells were placed at 37.degree. C. for at least 60 minutes before
placing them onto a rocking platform at 37.degree. C. Prior to
adding the cells, HILLEX.RTM.II (Solohill, MI) and Cytodex 3.RTM.
(GE Healthcare Life Sciences, NJ) micro-carriers were prepared as
directed by the manufacturer. Cells were grown in MEF-CM plus a
range of Rho kinase inhibitor concentrations, Y27632 at 10, 5, 2.5
or 1 .mu.M, or
(S)-(+)-4-Glycyl-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydr-
o-1H-1,4-diazepine dihydrochloride (Glycyl-H 1152 dihydrochloride
(H), Tocris, MO) at 5, 2.5, 1 or 0.5 .mu.M). The medium was changed
daily and one well of cells was counted at 4 and 7 days after
seeding for yield and viability (FIGS. 7, A and B). Overall, 10 or
5 .mu.M Y27632 (Sigma-Aldrich, MO) showed the best cell
proliferation (day 7) while 2.5 and 1.0 .mu.M appeared to have the
best attachment (day 4). Concentrations of 1 and 0.5 .mu.M Glycyl-H
1152 dihydrochloride (Tocris, MO) showed the best cell
proliferation (day 7) while 5 .mu.M appeared to have the best
attachment (day 4).
[0157] Next a dose titration of the Rho kinase inhibitor was
attempted since it has been characterized as a promoting apoptosis.
H1 cells at passage 48 were dissociated from MATRIGEL.TM. (BD
Biosciences, CA) coated plates with TrypLE.TM. Express. The cells
were then seeded onto 15 cm.sup.2 of micro-carriers into a single
well of a 12 well non-tissue culture treated plate. HILLEX.RTM.II
(Solohill, MI), Cytodex 1.RTM., or Cytodex 3.RTM. (GE Healthcare
Life Sciences, NJ) micro-carriers were tested with decreasing
amounts of Rho kinase inhibitor: 10 .mu.M Y27632 (Sigma-Aldrich,
MO) was used on day one followed by 0.5 .mu.M on day two (Y10/5
.mu.M); 2.5 .mu.M Glycyl-H 1152 dihydrochloride (Tocris, MO) was
used on day one followed by 0.5 .mu.M on day two (H2.5/0.5 .mu.M);
1 .mu.M Glycyl-H 1152 dihydrochloride (Tocris, MO) was used on day
one followed by 0.5 .mu.M Glycyl-H 1152 dihydrochloride (Tocris,
MO) on day two (H1/0.5 .mu.M); or continuous addition of 0.25 .mu.M
Glycyl-H 1152 dihydrochloride (Tocris, MO) was applied daily in
MEF-CM (H0.25 .mu.M). H1 cells and micro-carriers were agitated
every 45 minutes for 3 hours at 37.degree. C. before being placed
on a rocking platform at 37.degree. C. Cells were counted after 3,
5 and 7 days on the rocking platform at 37.degree. C. (FIG. 8).
Overall the best concentration of Glycyl-H 1152 dihydrochloride
(Tocris, MO) was 1-2.5 .mu.M on day one and 0.5 .mu.M on day 2
followed by withdrawal of the compound. The cells exhibited similar
growth rates at these concentrations of Glycyl-H 1152
dihydrochloride (Tocris, MO) compared to 10 .mu.M Y27632
(Sigma-Aldrich, MO) for all micro-carriers tested. Maintaining the
cells in 0.25 .mu.M Glycyl-H 1152 dihydrochloride (Tocris, MO)
resulted in poor cell yield. Using a minimal amount of Rho-kinase
inhibitor also helps reduce costs for the process and maybe
beneficial to cell proliferation. These data also show that human
embryonic stem cells did not require Rho kinase inhibitor in order
to remain attached to micro-carriers and to proliferate.
Example 3
Effect of Cell Density on Attachment and Growth on
Micro-Carriers
[0158] Improving the seeding density is a method to reduce the
total number of cells needed. To determine the proper seeding
density, the number of micro-carriers per 4.times. objective field
was counted. H1 cells were seeded at 0.4.times.10.sup.4
cells/cm.sup.2 (low), 1.2.times.10.sup.4 cells/cm.sup.2 (mid), or
3.times.10.sup.4 cells/cm.sup.2 (high) densities into a 10 cm plate
with Cytodex 3 micro-carriers (GE Healthcare Life Sciences, NJ) in
MEF-CM plus 10 .mu.M Y27632 (Sigma-Aldrich, MO). The plate was then
agitated every 45 minutes for 6 hours at 37.degree. C. The cells
and micro-carriers were transferred to a spinner flask (described
in Example 5) at 37.degree. C. at 30 rpm in 50 ml MEF-CM plus 10
.mu.M Y27632 (Sigma-Aldrich, MO). After 24 hours 25 ml of MEF-CM
with 5 .mu.M Y27632 (Sigma-Aldrich, MO) was added. After 24 hours
the speed of rotation was increased to 40 rpm. On day 3 and 5 of
culture, 50 ml of 75 ml was removed and replaced with MEF-CM.
Images were taken of an aliquot from the spinner flasks at 6 hours,
3 days, 5 days and 7 days. The percentage of micro-carriers with
cells attached is stated in the lower right corner in FIG. 9
images. At 3 days post seeding, the number of micro-carriers coated
with cells corresponds to the original seeding density but at days
5 and 7 the number of micro-carriers coated with cells did not
increase for the lower density seeded cells. This suggests that
0.4.times.10.sup.4 cells/cm.sup.2 is not a sufficient number of
cells to allow incorporation of micro-carriers into the aggregates.
At 3.times.10.sup.4 cells/cm.sup.2 the number of micro-carriers
with cells attached is similar to 1.2.times.10.sup.4 cells/cm.sup.2
seeded at days 5 and 7 (FIG. 9). When looking at the cell number,
it is clear that more cells are attached to micro-carriers from
high density cell seeding (FIG. 10). Analysis of the fold change
compared to the starting seeding cell number reveals a higher
number of cells attached at 3 and 5 days in the high density seeded
cultures (FIG. 11). By day 7, the control and high density seeded
cultures have similar fold change in cell number from their
starting seeding density. From these data, we conclude that
1.2.times.10.sup.4 cells/cm.sup.2 is the minimum cell number for
efficient attachment and growth of H1 cells on micro-carriers.
Moving to higher seeding densities may aid in decreasing the number
of days required for cell expansion.
Example 4
Dissociation of Cells from Micro-Carriers
[0159] In order to determine growth rates it was necessary to
dissociate the cells from the micro-carriers. Removal of the Rho
kinase inhibitor Y27632 (Sigma-Aldrich, MO) did not cause the H1
cells to dissociate from the micro-carriers (Example 2, FIG. 12).
H9 cells on HILLEX.RTM.II micro-carriers (Solohill, MI) were imaged
at 10.times. and 20.times. magnification before dissociation of the
cells from the micro-carriers (FIG. 13 A, B respectively).
Enzymatic treatment of the H9 cells on HILLEX.RTM.II micro-carriers
(Solohill, MI) allowed for detachment of viable cells (FIGS. 13 C,
D and 14). The H9 cells were grown for 6 days in a 6 well dish with
HILLEX.RTM.II micro-carriers (Solohill, MI) on a rocking platform
at 37.degree. C. The cells attached to micro-carriers were placed
in a 15 ml conical tube and the medium was aspirated after allowing
the micro-carriers to settle. The settled micro-carriers were
washed three times with 4 ml PBS (without magnesium and calcium
ions) allowing the micro-carriers to settle by gravity
sedimentation. The PBS was aspirated and 1 ml of PBS was added. The
micro-carriers with cells were transferred into a single well of a
12 well non-tissue culture treated plate. The plate was allowed to
rest at an angle to allow the micro-carriers to settle. The PBS was
aspirated and 1 ml TrypLE.TM. Express (Invitrogen, CA) or 0.05%
Trypsin/EDTA was added to the well. The plate was placed at
37.degree. C. on the rocking platform for 10 or 20 minutes. The
plate was removed and 3 ml DMEM/F12 or MEF-CM was added to the
well. The medium was vigorously pipetted, releasing the cells (FIG.
13 C, D). Observation of the micro-carriers under a microscope
determined detachment of the cells from the micro-carriers. The
cells were then centrifuged at 200 .mu.g for 5 minutes. The medium
was aspirated and the pellet was resuspended in 1 ml DMEM/F12 or
MEF-CM medium. The cells were then counted on a Guava PCA-96 (Guava
Technologies, Hayward, Calif.) with Viacount dye. Specifically, a
200 .mu.l volume of cells in appropriate dilution of medium, was
incubated with 2 .mu.l of Viacount for 10 minutes. The viability
and cell number were determined (FIG. 14). Both TrypLE.TM. Express
and Trypsin/EDTA dissociated the cells effectively from
micro-carriers.
[0160] Since TrypLE.TM. Express released the cells from the
micro-carriers and is available as a GMP product, it was tested
against other possible dissociation agents, specifically
Collagenase and Accutase.TM. (Sigma-Aldrich, MO). H1 p48 cells were
grown in a spinner flask (Example 5) for 10 days. The
micro-carriers were then collected and transferred to a 50 ml
conical tube. The cells were washed in PBS as above and transferred
to a 12 well plate. PBS was aspirated and 1 ml of TrypLE.TM.
Express, Accutase.TM. or Collagenase (10 mg/ml) was added to the
well and placed on a rocking platform at 37.degree. C. for 5 or 10
minutes. The cells/micro-carriers were vigorously resuspended in
DMEM/F12, and then the dissociated cells and micro-carriers were
passed through a 40 .mu.m cell strainer over a 50 ml conical tube.
The well was washed with an additional 2 ml medium, also added to
the strainer before centrifuging at 200.times.g for 5 minutes. The
cells were then resuspended in 1 ml DMEM/F12 and diluted for cell
counting, as above. Cell viability was similar with all tested
enzymes. Accutase.TM. and TrypLE.TM. Express released similar cell
numbers over 5 and 10 minute incubations (FIG. 15). This
illustrates the suitability of Accutase.TM. and TrypLE.TM. Express
as cell dissociation regents for human embryonic stem cells on
micro-carriers.
Example 5
Propagation of Undifferentiated Pluripotent Stem Cells on
Micro-Carriers
[0161] In order to expand cells on micro-carriers, cells must be
able to detach or be enzymatically dissociated from the
micro-carriers and reattach to new micro-carriers. Typical methods
of cell propagation on micro-carriers rely on the property of cells
to detach and reattach. The following experiment showed that this
was not a characteristic of human embryonic stem cells.
Specifically, H9 p43 cells were seeded onto HILLEX.RTM.II
micro-carriers (Solohill, MI) and incubated in a 125 ml spinner
flask (see below). Phenol red present in the medium and was taken
up by the HILLEX.RTM.II micro-carriers (Solohill, MI). After 8 days
of growth, a 10 ml aliquot of the cells on micro-carriers was
placed in a new spinner flask containing phenol red-free MEF-CM,
440 mg of HILLEX.RTM.II micro-carriers, and 5 .mu.M Y27632
(Sigma-Aldrich, MO). After 5 days incubation at 37.degree. C. with
30 rpm rotation, the micro-carriers were removed and images were
acquired (FIG. 16). The dark micro-carriers shown are the
micro-carriers covered with H9 cells grown in medium containing
phenol red. The light micro-carriers are the newly added
micro-carriers. It was expected that the H9 cells would detach and
reattach to new micro-carriers, however, instead the cells formed
aggregates with the new micro-carriers. No light micro-carriers had
cells attached that are not also in aggregates with the dark
micro-carriers, suggesting that the cells were not able to detach
and reattach to micro-carriers. In order to propagate cells grown
on micro-carriers, the cells must be enzymatically dissociated from
the micro-carriers (see Example 4).
[0162] Since it is now established how human embryonic stem cells
can be propagated on micro-carriers it needs to be determined how
human embryonic stem cells propagate in larger scale spinner
flasks. Spinner flasks allow the expansion of cells in high-density
systems. This is space conserving and is considered the first step
to expanding cells in bioreactors. To test the ability of human
embryonic stem cells to proliferate in spinner flasks, H9 passage
43 cells were seeded into 125 ml spinner flasks. Cells were
initially attached to the micro-carriers in a 10 cm plate before
transferring to the spinner flask. Specifically, H9 cells were
released from the two six-well dishes by a five minute incubation
with TrypLE.TM. Express at 37.degree. C. Prior to passaging with
TrypLE.TM. Express the cells had been passaged with Collagenase (1
mg/ml) and seeded onto 1:30 Growth Factor Reduced MATRIGEL.TM. (BD
Biosciences, CA) coated plates. The cells were resuspended in
DMEM/F12 and counted on a Guava instrument with Viacount. After
centrifugation, 3.times.10.sup.6 cells were seeded into a 10 cm
plate containing MEF-CM plus 10 .mu.M Y27632 (Sigma-Aldrich, MO)
and 250 cm.sup.2 of HILLEX.RTM. II micro-carriers (Solohill, MI),
prepared according to the manufacturer's instructions. The dish was
placed at 37.degree. C. and gently rotated and agitated once every
45 minutes for 4.5 hours. Then the cells, micro-carriers and medium
were transferred to a 125 ml spinner flask. The spinner flask was
then filled to 50 ml with MEF-CM plus 10 .mu.M Y27632
(Sigma-Aldrich, MO) and placed on a stir plate at 37.degree. C. at
40 rpm. The following day the medium was changed and filled to 75
ml with MEF-CM plus 5 .mu.M Y27632 (Sigma-Aldrich, MO). The rate of
stirring was increased to 70 rpm. Medium was changed every other
day without addition of Y27632 compound (Sigma-Aldrich, MO). The
cells were passaged according to the methods disclosed in Example
4, and 3.times.10.sup.6 cells were reseeded onto 250 cm.sup.2 of
new micro-carriers. The cultures were passaged when they reached a
confluency of 1-2.times.10.sup.5 cells/cm.sup.2. This was conducted
for 5 passages (FIG. 17). At each passage pluripotent marker
expression was evaluated showing 80-95% of cells expressed the
pluripotency markers CD9, SSEA4, SSEA3, TRA-1-60 and TRA-1-81 (FIG.
19A). A similar experiment was conducted with H9 p43 cells on
Cytodex 3.RTM. micro-carriers ((GE Healthcare Life Sciences, NJ),
FIG. 18, 19B). Overall, the cells proliferated well on both
HILLEX.RTM.II (Solohill, MI) and Cytodex 3.RTM. (GE Healthcare Life
Sciences, NJ) micro-carriers and remained pluripotent. Karyotypic
analysis was conducted after 5 passages in spinner flasks and
showed an abnormal trisomy in chromosome 12 in 1.5% of the cells.
Since these cells were nearing passage 50 at the conclusion of the
experiment, it may be a common occurrence to observe such
abnormalities. Beginning with a lower cell passage number may allow
this premise to be tested.
[0163] Similar experiments were conducted with the H1 line at p48
and p49. All parameters remained the same except the rotation speed
and seeding density. The rotation speed for the spinner flask was
30 rpm over-night on day 1 and increased to 40 rpm for all
additional days. The seeding density for Cytodex 3.RTM.
micro-carriers (GE Healthcare Life Sciences, NJ) was about 11,000
cells/cm.sup.2 while the seeding density for Cytodex 1.RTM.
micro-carriers (GE Healthcare Life Sciences, NJ) was about 7,000
cells/cm.sup.2. The cell number seeded was held constant at
3.times.10.sup.6 cells per spinner flask. The weight of
micro-carriers was held constant at 100 mg for Cytodex 1.RTM. and
Cytodex 3.RTM.. One advantage of Cytodex 1.RTM. and Cytodex 3.RTM.
over HILLEX.RTM.II micro-carriers is their larger surface area.
FIGS. 20 and 21 show the expansion of H1 cells on Cytodex 1.RTM.
and Cytodex 3.RTM. respectively. The cells remained pluripotent
over the five passages (FIG. 22). Karyotype analysis of H1 cells on
Cytodex 3.RTM. micro-carriers revealed duplication of the Y
chromosome in 10% of the cells tested. These H1 cells were passaged
onto micro-carriers at p48 and were analyzed 5 passages later.
H1p55 cells grown on MATRIGEL.TM. (BD Biosciences, CA) on a planar
surface had a normal karyotype. Analysis of the doubling rates for
these cells between day 3 and the day of passaging (day 5, 6 or 7)
showed no overall change in doubling times (FIG. 23). H1 cells
grown on Cytodex 1.RTM. micro-carriers and H9 grown on
HILLEX.RTM.II micro-carriers (Solohill, MI) showed the most
consistent doubling times (Table 3).
Example 6
Proliferation of Human Embryonic Stem Cells on Micro-Carriers in
Defined Medium
[0164] To manufacture a therapeutic product, it is desirable to
remove any animal components from the human embryonic stem cell
culture medium. Currently human embryonic stem cells are maintained
on MATRIGEL.TM. (BD Biosciences, CA) in medium conditioned using
mouse embryonic fibroblasts (MEF-CM). Both MATRIGEL.TM. (BD
Biosciences, CA) and MEF-CM are derived from mouse cells.
Additionally, MEF-CM is an expensive and time-consuming medium to
generate. To determine if human embryonic stem cells can be
sustained on micro-carriers with defined medium, H9 cells were
seeded onto Cytodex 3.RTM. (GE Healthcare Life Sciences, NJ) and
HILLEX.RTM.II (Solohill, MI) micro-carriers in the presence of Rho
kinase inhibitors, 10 .mu.M Y27632 (Sigma-Aldrich, MO) or 2.5 .mu.M
Glycyl-H 1152 dihydrochloride (Tocris, MO) in Stem Pro (Invitrogen,
CA), mTESR (StemCell Technologies, Vancouver, Canada) or MEF-CM.
The cells were placed in a 12 well dish on a rocking platform at
37.degree. C. The cells were counted at days 3, 5 and 7. H9 p39
cells grown in MEF-CM on both bead types showed typical expansion
characteristics (FIG. 24). Similar cells grown in mTESR (StemCell
Technologies, Vancouver, Canada) proliferated well on Cytodex
3.RTM. micro-carriers in the presence of 10 .mu.M Y27632
(Sigma-Aldrich, MO) but exhibited a slow growth rate on
HILLEX.RTM.II micro-carriers. Cells of the human embryonic stem
cell line H9 at passage 64 (H9p64) cells that had been acclimated
to StemPro medium for over 20 passages proliferated well on both
HILLEX.RTM.II and Cytodex 3.RTM. in the presence of 10 .mu.M Y27632
(Sigma-Aldrich, MO). Surprisingly, these cells did not proliferate
well in the presence of 2.5 .mu.M Glycyl-H 1152 dihydrochloride
(Tocris, MO) on Cytodex 3.RTM. micro-carriers. Therefore the
micro-carrier type, Rho kinase inhibitor, and medium all play a
role in determining the ability of human embryonic stem cells to
proliferate.
[0165] H1 human embryonic stem cells at passage 38 were seeded onto
either Cytodex 3.RTM. or HILLEX.RTM.II micro-carriers in the
presence of Rho kinase inhibitors, 10 .mu.M Y27632 (Sigma-Aldrich,
MO) or 2.5 .mu.M Glycyl-H 1152 dihydrochloride in mTESR (StemCell
Technologies, Vancouver, Canada) or MEF-CM in a 12 well dish. The
cells were placed on a rocking platform at 37.degree. C. The cells
were counted at days 3, 5 and 7. Cells grown in MEF-CM on both
micro-carrier types showed typical expansion characteristics in the
presence of Y27632 (Sigma-Aldrich, MO) but exhibited poor growth
with Glycyl-H 1152 dihydrochloride (Tocris, MO) on Cytodex 3.RTM.
(FIG. 25). mTESR medium (StemCell Technologies, Vancouver, Canada)
allowed the H1 cells to proliferate on HILLEX.RTM.II micro-carriers
in the presence of both Rho kinase inhibitors but exhibited low
growth rate on Cytodex 3.RTM. micro-carriers.
[0166] Given that H1 p50 cells proliferated well in mTESR (StemCell
Technologies, Vancouver, Canada) on HILLEX.RTM.II micro-carriers,
3.times.10.sup.6 cells were seeded onto 250 cm.sup.2 HILLEX.RTM.II
micro-carriers. Cells were incubated at 37.degree. C. in a 10
cm.sup.2 dish for 5 hours with agitation by hand every 45 minutes.
mTESR (StemCell Technologies, Vancouver, Canada) plus 10 .mu.M
Y27632 (Sigma-Aldrich, MO) was changed every other day. This was
conducted in parallel with cells grown in MEF-CM (Example 5).
Unlike cells grown in MEF-CM, the cells grown in mTESR medium
(StemCell Technologies, Vancouver, Canada) began to detach from the
HILLEX.RTM.II micro-carriers after 7 days (FIG. 26A vs. 26B). This
indicates that additional supplements needed to be added to mTESR
(StemCell Technologies, Vancouver, Canada) in order for the human
embryonic stem cells to remain attached and proliferate on
HILLEX.RTM.II micro-carriers (Solohill, MI).
Example 7
Differentiation of Human Embryonic Stem Cells on Micro-Carriers
[0167] Since the human embryonic stem cells can be expanded on
micro-carriers, the differentiation potential of these cells must
be determined. Cells of the human embryonic stem cell line H9 at
passage 43 were passaged five times on Cytodex 3.RTM.
micro-carriers (GE Healthcare Life Sciences, NJ). At passage 5, the
cells were grown for 6 days on the micro-carriers before being
dissociated from the micro-carriers with TrypLE.TM. Express (see
Example 4). The cells were then plated on 1:30 MATRIGEL.TM.:
DMEM/F12 coated plates. After the cells became 80 to 90% confluent
on the plates they were exposed to differentiating agents.
Differentiation of the human embryonic stem cells to definitive
endoderm was conducted by treating the cells for 2 days with 2%
Albumin Bovine Fraction V Fatty Acid Free (FAF BSA, MP Biomedicals,
Ohio) in RPMI plus 100 ng/ml Activin A (PeproTech, NJ), 20 ng/ml
Wnt3a (R&D Biosciences, MN) and 8 ng/ml bFGF (PeproTech, NJ).
The cells were treated for an additional 2 days in 2% FAF BSA in
RPMI plus 100 ng/ml Activin A (PeproTech, NJ) and 8 ng/ml bFGF
(PeproTech, NJ). Medium was changed daily. FACS analysis conducted
for the definitive endoderm cell surface marker CXCR4, showed that
87% of the cells expressed the protein (FIG. 27A). A similar
experiment was conducted with cells of the human embryonic stem
cell line H1 at passage 49 grown on Cytodex 1.RTM. micro-carriers
(GE Healthcare Life Sciences, NJ) for 5 passages, revealing that
91% of the cells differentiated on the micro-carriers expressed
CXCR4 (FIG. 27B). This demonstrates that the cells grown on
micro-carriers are capable of differentiating into definitive
endoderm, the first step to becoming insulin-producing cells.
[0168] Three types of micro-carriers, Cytodex 1.RTM., Cytodex
3.RTM. (GE Healthcare Life Sciences, NJ) and HILLEX.RTM.II
(Solohill, MI), allow attachment and growth of H1 cells.
Differentiation of H1 cells on these three micro-carriers was
conducted. The cells were grown on these micro-carriers in spinner
flasks (Example 5) for various passage numbers (1 to 5). Six to
eight days after the last passage aliquots of the micro-carriers
plus cells in suspension were transferred to 6 or 12 well plates. A
total of 15 cm.sup.2 of micro-carriers plus cells per 12 well plate
well or 30 cm.sup.2 micro-carriers plus cells per 6 well plate was
transferred. Differentiation medium was then added to the plate
wells and the plate was placed on a rocking platform at 37.degree.
C. Differentiation of the human embryonic stem cells to definitive
endoderm was conducted by treating the cells for 2 days with 2%
Albumin Bovine Fraction V Fatty Acid Free (MP Biomedicals, Ohio) in
RPMI plus 100 ng/ml Activin A (PeproTech, NJ), 20 ng/ml Wnt3a
(R&D Biosciences, MN) and 8 ng/ml bFGF (PeproTech, NJ). The
cells were treated for an additional 2 days in 2% FAF BSA in RPMI
plus 100 ng/ml Activin A (PeproTech, NJ) and 8 ng/ml bFGF
(PeproTech, NJ). Medium was changed daily. FACS analysis was
conducted for the definitive endoderm cell surface marker CXCR4
(FIG. 28). Cells grown on Cytodex 1.RTM., and Cytodex 3.RTM.
micro-carriers supported differentiation to definitive endoderm
(87% and 92% respectively while HILLEX.RTM.II micro-carriers did
not support differentiation as to the same extent as the other
micro-carriers tested in this experiment (42%).
[0169] To determine if the cell density affects differentiation of
the cells on micro-carriers, cells of the human embryonic stem cell
line H1 at passage 40 were grown on Cytodex 3.RTM. micro-carriers
in a spinner flask for either 8 days or 11 days. Then the
equivalent of about 15 cm.sup.2 of micro-carriers plus cells was
placed in a 6 well dish and placed on a rocking platform. The cells
were then incubated in definitive endoderm differentiating medium
as above. After 4 days the cells were analyzed by FACS for CXCR4
expression. 87% of the cells grown for 6 days in spinner flask
expressed CXCR4 while 56% of cells grown for 11 days in the spinner
flask expressed CXCR4 (FIG. 29). This demonstrates that the number
of days that the cells are in culture is important prior to
differentiation, specifically, if the cell density is too high it
may not allow the cells to efficiently differentiate.
[0170] To determine if human embryonic stem cells could be
differentiated into pancreatic endoderm cells on all three
micro-carrier types determined sufficient for attachment and
growth, cells of the human embryonic stem cell line H1 at passage
41 (H1p41) were seeded on to Cytodex 1.RTM., Cytodex 3.RTM.
micro-carriers (GE Healthcare Life Sciences, NJ) and HILLEX.RTM.II
micro-carriers (Solohill, MI) (see Example 1). Micro-carriers were
prepared according to the manufactures instructions. 30 cm.sup.2 of
micro-carriers were transferred to low attachment 6 well plates.
The H1 cells were dissociated from two 10 cm.sup.2 plates with
TrypLE.TM. Express according to manufacturer's instructions. Cell
were seeded at 5.times.10.sup.5 cells per well. Attachment of the
cells to the beads was carried out according to the methods
described in Example 3. Briefly, the cells and micro-carriers were
incubated in MEF conditioned media with 10 .mu.M Y27632 at
37.degree. C. for four hours with brief agitation each hour. The
cells on HILLEX.RTM.II and Cytodex 10 micro-carriers were placed on
a rocking platform. The cells on Cytodex 3.RTM. micro-carriers were
allowed to sit undisturbed overnight. The media was changed daily
and no longer included Y27632.
[0171] Due to poor attachment in this experiment, the majority of
cells in the Cytodex 1.RTM. plate were no longer attached to the
micro-carriers. However, longer attachment time and/or slower
rocking speed may improve the cell attachment. After 7 days, the
cells were differentiated to definitive endoderm with 2% fatty acid
free (FAF) BSA (Proliant, IA) in RPMI and the following growth
factors: bFGF (8 ng/ml, (PeproTech, NJ)), Activin A (100 ng/ml,
(PeproTech, NJ)), Wnt3a (20 ng/ml, (R&D Biosciences, MN)). For
the second through fourth day of differentiation, the cells were
treated with the same media lacking Wnt3a. FACS analysis of
duplicate samples after 4 days revealed CXCR4 levels of 77-83%
positive cells. Definitive endoderm expression was equivalent
between cells grown on the different micro-carriers. See FIG.
30.
[0172] The cells were then differentiated further for 2 days with
FGF7 (50 ng/ml, (R&D Systems, MN)), KAAD-Cyclopamine (0.25
.mu.M, (Calbiochem, NJ)) in DMEM/F12 or DMEM-HG plus 2% FAF BSA
(Proliant, IA). This was followed by four days of treatment with
Noggin (100 ng/ml, (R&D Biosciences, MN)), FGF7 (50 ng/ml,
(R&D Systems, MN)), Retinoic Acid (2 .mu.M, (Sigma-Aldrich,
MO)), and KAAD-Cyclopamine (0.25 .mu.M, (Calbiochem, NJ)) in
DMEM/F12 or DMEM-HG with 1% B-27 supplement (Invitrogen, CA). The
cells were then differentiated for three days with Noggin (100
ng/ml, (R&D Biosciences, MN)), DAPT (1 .mu.M, (Sigma-Aldrich,
MO)), Alk5 inhibitor II (1 .mu.M, (Axxora, CA)) in DMEM/F12 or
DMEM-HG with 1% B-27 supplement (day 13, pancreatic endoderm,
(Invitrogen, CA)). FIG. 31 shows the expression level by Q-PCR for
the pancreatic specific genes, NKX6.1, PDX1 and NGN3. CT values
clearly show that cells differentiated on HILLEX.RTM.II
micro-carriers do not differentiate efficiently to express the
necessary beta cell precursor cell markers. Although the cells
differentiated efficiently to definitive endoderm on all three
micro-carrier types further differentiation to pancreatic
progenitors is not efficient on HILLEX.RTM.II micro-carriers.
[0173] To determine if the human embryonic stem cells could be
further differentiated into insulin producing cells, H1 p45 cells
were grown on Cytodex 3.RTM. micro-carriers (GE Healthcare Life
Sciences, NJ) and differentiated similar to above. Briefly, H1
cells were dissociated from 10 cm.sup.2 plates with TrypLE.TM.
Express according to manufacturer's instructions. Cells were seeded
at 1 or 2.times.10.sup.6 cells per 6 well plate well. Attachment of
the cells to the beads is described in Example 3. Briefly, the
cells and micro-carriers were incubated in MEF conditioned media
with 10 .mu.M Y27632 at 37.degree. C. for four hours with brief
agitation each hour. The cells were then allowed to incubate
overnight undisturbed. On day 2 the media was replaced with MEF
conditioned media plus 5 uM Y27632 and the plates were placed on a
rocking platform. The media was changed each subsequent day without
Y27632. On day five, the media was replaced with definitive
endoderm differentiation media, 2% fatty acid free (FAF) BSA
(Proliant, IA) in RPMI with the following growth factors: bFGF (8
ng/ml, (PeproTech, NJ)), Activin A (100 ng/ml, (PeproTech, NJ)),
Wnt3a (20 ng/ml, (R&D Biosciences, MN)). For the second and
third day of differentiation, the cells were treated with the same
media lacking Wnt3a. FACS analysis of duplicate samples after 3
days revealed CXCR4 levels of 97-98% positive cells. The cells were
then differentiated further for 2 days with FGF7 (50 ng/ml,
(R&D Systems, MN)), KAAD-Cyclopamine (0.25 .mu.M, (Calbiochem,
NJ)) in DMEM-High Glucose (HG) plus 2% FAF BSA (Proliant, IA). This
was followed by four days of treatment with Noggin (100 ng/ml,
(R&D Biosciences, MN)), FGF7 (50 ng/ml, (R&D Systems, MN)),
Retinoic Acid (2 .mu.M, (Sigma-Aldrich, MO)), and KAAD-Cyclopamine
(0.25 .mu.M, (Calbiochem, NJ)) in DMEM-HG with 1% B-27 supplement
(Invitrogen, CA). The cells were then differentiated for three days
with Noggin (100 ng/ml, (R&D Biosciences, MN)), DAPT (1 .mu.M,
(Sigma-Aldrich, MO)), Alk5 inhibitor II (1 .mu.M, (Axxora, CA)) in
DMEM-HG or DMEM-F12 with 1% B-27 supplement (Invitrogen, CA). This
was followed by differentiation in DMEM-HG or DMEM-F12 with Alk5
inhibitor II (1 .mu.M, (Axxora, CA)) for seven days. Final
differentiation was for five days in DMEM-HG or DMEM-F12
respectively. This is a total of 24 days of differentiation leading
to expression of pancreatic endocrine hormones. FIG. 32 shows the
FACS analysis results of the cells at this end point. Cells with
the highest seeding density and differentiated in DMEM-HG from days
6 through 24 had the highest levels of insulin expression (FIG.
32).
[0174] Alternatively, to determine if the human embryonic stem
cells could be differentiated into insulin producing cells, H1 p44
cells were grown in a spinner flask for 7 days on Cytodex 3.RTM.
micro-carriers (see Example 5). The cells plus micro-carriers were
transferred to a 12 well plate at 15 cm.sup.2/well and placed on a
rocking platform at 37.degree. C. The cells were differentiated to
definitive endoderm as above but with DMEM/F12 instead of RMPI.
FACS analysis after 4 days revealed CXCR4 levels of 75 to 77%
positive cells in a triplicate analysis. The cells were then
differentiated further with 3 days of treatment with FGF7 (50
ng/ml, (R&D Systems, MN)), KAAD-Cyclopamine (0.25 .mu.M,
(Calbiochem, NJ)) in DMEM/F12 plus 2% Albumin Bovine Fraction V
Fatty Acid Free. This was followed by four days of treatment with
Noggin (100 ng/ml, (R&D Biosciences, MN)), FGF7 (50 ng/ml,
(R&D Systems, MN)), Retinoic Acid (2 .mu.M, (Sigma-Aldrich,
MO)), and KAAD-Cyclopamine (0.25 .mu.M, (Calbiochem, NJ)) in
DMEM/F12 with 1% B-27 supplement (Invitrogen, CA). The cells were
then differentiated for three days with Noggin (100 ng/ml, (R&D
Biosciences, MN)), Netrin4 (100 ng/ml, (R&D Biosciences, MN)),
DAPT (1 .mu.M, (Sigma-Aldrich, MO)), Alk5 inhibitor II (1 .mu.M,
(Axxora, CA)) in DMEM/F12 with 1% B-27 supplement (day 15,
pancreatic endoderm, (Invitrogen, CA)). This was followed by six
days of treatment with Alk5 inhibitor II (1 .mu.M, (Axxora, CA)) in
DMEM/F12 with 1% B-27 supplement (day 21, pancreatic endocrine
cells, (Invitrogen, CA)). The final treatment for seven days was
DMEM/F12 with 1% B-27 supplement (day 28, insulin-expressing cells,
(Invitrogen, CA)). FIG. 33 shows the expression level by Q-PCR for
the pancreatic specific genes, insulin, Pdx1 and glucagon. The data
on micro-carriers is compared to previous data of H1 p42 cells
differentiated on a MATRIGEL.TM. (BD Biosciences, CA) coated planar
surface. The expression level of these pancreas specific genes is
similar or better for micro-carrier differentiated cells compared
to cells differentiated on planar surfaces.
[0175] Similar experiments were conducted with H9p38 cells passaged
onto Cytodex 3.RTM. micro-carriers and expanded in a spinner flask.
An aliquot of 15 cm.sup.2 of micro-carriers plus cells was placed
in a 12 well plate and placed on a rocking platform with
differentiation medium. This was compared to cells plated on a 6
well plate coated with MATRIGEL.TM. (BD Biosciences, CA).
Differentiation of the cells to definitive endoderm in RPMI and
supplements was achieved, with an average of 83% of the cells
expressing CXCR4 (samples in duplicate) compared to 72% of cells
expressing CXCR4 on a planar substrate (FIG. 34). Further
differentiation to pancreatic endoderm (day 15),
pancreatic-endocrine cells (day 22) and insulin-expressing cells
(day 29) showed similar expression levels of insulin and glucagon
between cells grown on micro-carriers to those grown on a planar
substrate (FIG. 35). The medium components were identical to those
listed for the above H1 differentiation experiment with one
additional day in the endocrine cell differentiating components. At
the insulin-expressing stage, cells showed a surprising decrease in
insulin expression compared to day 22. Since the decrease was noted
in both micro-carrier and planar samples, it is likely not due to
the attachment substrate. This shows that H9 cells can also be
successfully differentiated to at least pancreatic endocrine cells
on micro-carriers.
[0176] Overall, two different human embryonic stem cell lines, H1
and H9, can be differentiated to pancreatic endocrine cells on
Cytodex 3.RTM. micro-carriers, illustrating the potential to expand
and differentiate these cells in a large-scale culture system
(FIGS. 17, 21, 33, and 35). Human embryonic stem cells were able to
attach and proliferate to at least three micro-carrier bead types
and the cells could be differentiated to at least definitive
endoderm (FIG. 28). These results illustrate a method by which
human embryonic stem cells can be expanded and differentiated for
therapeutic uses.
Example 8
Human Embryonic Stem Cells Passaged as Single Cells in a 3D
Micro-Carrier Based Culture can be Transferred to Culture on an ECM
Free Surface while Maintaining Pluripotency
[0177] H1 human embryonic stem cells were cultured on
micro-carriers according to the methods described in Example 5.
Cells were removed from micro-carriers and plated to Nunc4, Nunc13,
CELLBIND.TM., or PRIMARIA.TM. tissue culture polystyrene (TCPS)
planar surfaces with MEFCM16 supplemented with 3 .mu.M Glycyl-H
1152 dihydrochloride. The cells were seeded at a density of 100,000
cells/cm.sup.2 in six well plates and then cultured for one
additional passage on the respective surface. Cells were then
either lifted with TrypLE and tested by flow cytometry for
pluripotency markers, or lysed in the well with RLT for mRNA
purification and qRT-PCR, or differentiated to definitive endoderm.
Differentiation was induced by treating the cells with RPMI media
supplemented with 2% BSA, 100 ng/ml Activin A, 20 ng/ml Wnt3a, 8
ng/ml bFGF, and 3 .mu.M Glycyl-H 1152 dihydrochloride for 24 hours.
Media was then changed to RPMI media supplemented with 2% BSA, 100
ng/ml Activin A, 8 ng/ml bFGF, and 3 .mu.M Glycyl-H 1152
dihydrochloride for an additional 48 hours with daily media
change.
[0178] As measured by pluripotency markers, using either flow
cytometry or qRT-PCR, cells cultured on micro-carriers and
transferred to culture on Nunc4, Nunc13, CellBIND, or Primaria
tissue culture polystyrene (TCPS) planar surfaces maintained
pluripotency after two passages on the respective planar surface
(FIG. 36). Furthermore, the cells maintained the capacity to
differentiate to a definitive endoderm fate as measured by either
flow cytometry or qRT-PCR (FIG. 37). Similar results were also
obtained in side-by-side tests of H1 and H9 human embryonic stem
cells passaged on Cytodex 3.RTM. micro-carriers and differentiated
to definitive endoderm (FIG. 38).
[0179] These results indicate that human embryonic stem cells can
be passaged on micro-carriers and then subsequently cultured on
another surface while maintaining pluripotency. The cells may also
be transferred to another surface and efficiently induced to
differentiate.
Example 9
Human Embryonic Stem Cells can be Transferred Directly from a
Cluster/Colony Style Culture on Mitotically Inactivated Fibroblast
Feeders to Culture as Single Cells on ECM Free Surfaces for at
Least 10 Passages without Loss of Pluripotency and without Manual
Removal of Fibroblast Feeders
[0180] Human embryonic stem cell lines are currently derived using
a method that promotes a colony outgrowth of a single cell or a
cluster of a few cells from a blastocyst. This colony outgrowth is
then serially passaged and propagated until enough cluster/colonies
of cells are available that they constitute a cell line. Once a
cell line has been derived, in order to maintain the pluripotent
and karyotypically stable characteristics of human embryonic stem
cells, the current standard in the art for high quality,
reproducible culture of human embryonic stem cells is to maintain
the clusters/colonies of human embryonic stem cells on a feeder
layer of mitotically inactive fibroblasts and to pass the cells
using manual disruption or gentle enzymatic bulk passage with
collagenase or neutral protease or a blend thereof. These passage
methods maintain human embryonic stem cells clusters and promote
colony style growth of human embryonic stem cell. After a stable
human embryonic stem cell line is established the cells can be
transitioned to an extracellular matrix (ECM) substrate such as
MATRIGEL.TM.. However, whether the cells are grown on fibroblast
feeders or on an ECM substrate, the recommended passage method for
human embryonic stem cells specifically instructs technicians not
to fully dissociate human embryonic stem colonies.
[0181] The current best practice for large scale culture of
mammalian cells is to use a 3-dimensional culture vessel that
tightly maintains homeostatic, uniform conditions and can
incorporate micro-carriers for support of adhesion dependent cells.
However, the current standard methods used for human embryonic stem
cell culture-growth on fibroblast feeders or an ECM substrate and
cluster/colony style culture pose a technical hurdle to
successfully growing and maintaining a pluripotent human embryonic
stem cell culture on micro-carriers, since these methods are not
easily transferable to large scale culture on micro-carriers. In
order to effectively grow human embryonic stem cells on
micro-carriers the human embryonic stem cell culture must be able
to be passaged as single cells, and not as colonies or clusters, as
is currently the standard in the art. Furthermore, the human
embryonic stem cells should be able to grow without a layer of
feeder cells or ECM substrate.
[0182] We describe below a method which addresses these technical
hurdles. We demonstrate how to convert human embryonic stem cell
cultures from clusters/colonies on a mitotically inactive
fibroblast feeder layer directly to a single cell culture system
that does not require an underlying fibroblast feeder layer or a
surface coated with MATRIGEL or other an extracellular matrix
substrate. This method utilizes bulk passage of human embryonic
stem without any manual removal of fibroblast feeder cells or
selection of pluripotent cells from the total cell population to
convert the culture directly from colony style, fibroblast feeder
based culture to feeder free/matrix free culture on PRIMARIA in the
presence of the Rho Kinase (ROCK) inhibitor, Glycyl-H 1152
dihydrochloride. This method can be completed in a sealed vessel to
adhere to regulatory requirements and produces a highly homogeneous
human embryonic stem culture that retains pluripotency and the
ability to differentiate to definitive endoderm, and does not
contain a fibroblast cell population.
[0183] Method: Cells were routinely passaged by aspirating media,
washing with PBS, and then treating the cells with a dissociation
enzyme (collagenase, Accutase.TM., or TrypLE). Collagenase was used
at 1 mg/ml concentration; Accutase.TM. or TrypLE were used at
1.times. stock concentration. All enzymes were used after reaching
room temperature. A solution of 2% BSA in DMEM/F12 was added to
each well and cells were uniformly suspended in the solution after
treating the cells with enzyme. Cells were then centrifuged for 5
minutes at 200 g, the cell pellet and additional 2% BSA in DMEM/F12
solution was added to resuspend cells and the cell suspension was
distributed to three 50 ml sterile conical tubes and centrifuged
for 5 min at 200 g.
[0184] Using a sequential method, we removed the fibroblast feeders
by high density passaging the cluster/colony style human embryonic
stem cells to a Primaria surface by treating the MEF based culture
with either Accutase.TM., TrypLE.TM., or collagenase. At the first
passage, cells were plated to T-25 flasks coated with a 1:30
dilution of MATRIGEL.TM. in mouse embryonic fibroblast (MEF)
conditioned media (CM) or the cells were plated to T-25
PRIMARIA.TM. culture flasks in MEF-CM plus 3 uM Glycyl-H 1152
dihydrochloride. All cells were plated at a split ratio of 1 to 3.5
and cells were exposed to enzyme for 10 minutes. Cell number for
cells lifted with TrypLE.TM. or Accutase.TM. was determined by
counting trypan blue stained cells with a hemocytometer. After
plating the cells the media was changed daily, and cells plated in
MEF-CM+3 .mu.M Glycyl-H 1152 dihydrochloride were fed daily with
MEF-CM+1 .mu.M Glycyl-H 1152 dihydrochloride and samples were
assayed for expression of mRNA markers of pluripotency and
differentiation. hESCs passaged twice as single cells under Matrix
free conditions maintained gene expression of pluripotency genes
and inhibited expression of differentiation genes (FIG. 39).
[0185] 2.sup.nd passage: Cells were passaged at a ratio of 1 to 4
using a 10 minutes exposure to TrypLE.TM. or Accutase.TM.. We also
introduced a shorter enzyme exposure time that was determined
empirically by treating the cells and monitoring for detachment. We
observed that 3 minute exposure to TrypLE.TM. and 5 minute exposure
to Accutase.TM. was sufficient to lift the cells. After treating
the cells with enzyme, the cells were passaged as described above
and aliquots of cell mRNA were taken for qRT-PCR at the time of
passaging.
[0186] 3.sup.rd passage: Upon reaching confluence cells were washed
with PBS, disrupted with enzyme for 3 or 10 minutes (TrypLE.TM.) or
5 or 10 minutes (Accutase.TM.), suspended in 2% BSA in DMEM/F12 and
centrifuged, washed again with 2% BSA in DMEM/F12, centrifuged, and
then resuspended and plated in their respective media. At this
passage cells were plated at 1:4 ratio and also at 2 additional
split ratios-1:8 and 1:16. Aliquots of cell mRNA were taken for
qRT-PCR at each passage.
[0187] 4 passages+: The conditions adopted for time of exposure to
enzyme and passage ratio at passages 2 and 3 were maintained from
passage 4 onward. Each time the culture grew to confluence cells
were washed with PBS, disrupted with enzyme for the specified time,
suspended in 2% BSA in DMEM/F12 and centrifuged, washed again with
2% BSA in DMEM/F12, centrifuged, and then resuspended in their
respective media at the specified plating ratio. The media for
cells plated to PRIMARIA was supplemented with 3 .mu.M Glycyl-H
1152 dihydrochloride at the time of plating. After plating, media
was changed daily and cells plated in MEF-CM+3 .mu.M Glycyl-H 1152
dihydrochloride were fed daily with MEF-CM+1.mu. Glycyl-H 1152
dihydrochloride. Aliquots of cell mRNA were taken for qRT-PCR at
the time of passaging.
[0188] At the completion of greater than 8 passages, cells were
assayed for pluripotency by flow cytometry for pluripotency surface
markers (FIG. 40) and by qRT-PCR for pluripotency and
differentiation markers (FIGS. 41, 42, and 43). Cells were also
differentiated to definitive endoderm by treating the cells with
RPMI media supplemented with 2% BSA, 100 ng/ml Activin A, 20 ng/ml
Wnt3a, 8 ng/ml bFGF, and 3 uM Glycyl-H 1152 dihydrochloride for 24
hours. Media was then changed to RPMI media supplemented with 2%
BSA, 100 ng/ml Activin A, 8 ng/ml bFGF, and 3 .mu.M Glycyl-H 1152
dihydrochloride for an additional 48 hours with daily media change.
Samples differentiated to definitive endoderm were then tested for
the presence of the definitive endoderm marker CXCR4 by flow
cytometry (FIG. 40).
[0189] These results indicate that bulk passage from colony style,
fibroblast feeder based culture to feeder free/matrix free culture
on PRIMARIA in the presence of the Rho Kinase (ROCK) inhibitor,
Glycyl-H 1152 dihydrochloride results in a highly homogeneous human
embryonic stem cell culture that retains pluripotency and the
ability to differentiate to definitive endoderm, and does not
contain a fibroblast cell population.
Example 10
Human Embryonic Stem Cells Transferred from Tissue Culture Plastic
to Micro-Carriers
[0190] H1 cells were cultured on PRIMARIA.TM. (cat. no. 353846,
Becton Dickinson, Franklin Lakes, N.J.) tissue culture plates
(method in Example 9) and released by treatment with TrypLE.TM.
Express for 3-5 minutes and seeded into 6 well non-tissue culture
treated plates with Cytodex 3.RTM. (GE Healthcare Life Sciences,
NJ) or HILLEX.RTM.II (Solohill, MI) micro-carriers in MEF-CM plus
10 .mu.M Y27632 (Sigma-Aldrich, MO). As a control, H1p46 cells
grown on MATRIGEL (BD Biosciences, CA) coated plates and passaged
with Collagenase (1 mg/ml) were released and seeded onto
micro-carriers in a similar manner. The plates were incubated at
37.degree. C. for 5 hours, agitating by hand every 45 minutes. The
plates were then placed on a rocking platform at 37.degree. C.
Medium was changed every day with MEF-CM plus 10 .mu.M Y27632
(Sigma-Aldrich, MO). Images show good attachment of cells to the
micro-carriers at 3 days (FIG. 44). After 7 days the cells were
released (described in Example 4 infra) and analyzed by FACS for
the pluripotency markers CD9, SSEA-4, SSEA-3, TRA-1-60, TRA-1-81
(FIG. 45). The majority of pluripotency markers were expressed on
90-100% of the cells. There are no clear differences between cells
passaged with Accutase.TM. (Millipore, MA) and TrypLE.TM. Express
(Invitrogen, CA) nor between growth on with Cytodex 3.RTM. (GE
Healthcare Life Sciences, NJ) and HILLEX.RTM.II (Solohill, MI)
micro-carriers. Overall, the cells remained pluripotent when
transferred from PRIMARIA.TM. (cat. no. 353846, Becton Dickinson,
Franklin Lakes, N.J.) cell culture plastic onto micro-carriers.
[0191] Next these H1 cells on the micro-carriers were
differentiated to definitive endoderm. The method is described in
Example 7. After 4 days of differentiation, the H1 cells were
released from the micro-carriers and underwent FACS analysis
showing greater than 82% of the cells expressing CXCR4. See FIG.
46. The cells were efficiently differentiated into definitive
endoderm regardless of the micro-carrier type or passaging enzyme
on PRIMARIA.TM. (cat. no. 353846, Becton Dickinson, Franklin Lakes,
N.J.). This proves the flexibility of the expansion system and
allows for cells to be grown without matrix on plastic and
micro-carriers.
Example 11
Human Embryonic Stem Cells Transferred from Planar Substrates
Consisting of Mixed Cellulose Esters to Micro-Carriers
[0192] H1 cells were cultured on planar substrates consisting of
mixed cellulose esters for 12 passages, according to the methods
disclosed U.S. Patent Application No. 61/116,452. The cells were
released from the planar substrate by treatment with TrypLE.TM.
Express for 3-5 minutes and seeded into 6 well non-tissue culture
treated plates with CYTODEX 3.RTM. (GE Healthcare Life Sciences,
NJ) or HILLEX.RTM.II (Solohill, MI) micro-carriers in MEF-CM plus
10 mM Y27632 (Sigma-Aldrich, MO). As a control, H1p44 cells grown
on MATRIGEL.TM. coated plates (BD Biosciences, CA), passaged with
Collagenase (1 mg/ml) were released and seeded onto micro-carriers
in a similar manner. The plates were incubated at 37.degree. C. for
5 hours, agitating by hand every 45 minutes. The plates were then
placed on a rocking platform at 37.degree. C. Media was changed
daily. After 7 days the cells were released (described in Example
4) and analyzed by FACS for the pluripotency markers CD9, SSEA-4,
SSEA-3, TRA-1-60, TRA-1-81 (FIG. 47). The majority of pluripotency
markers were expressed on greater than 90% of the cells. There were
no clear differences between cells grown on CYTODEX 3.RTM. and
HILLEX.RTM.II micro-carriers. H1p44 control cells were not tested
for pluripotency after growth on HILLEX.RTM.II micro-carriers,
since pluripotency had been confirmed by other experiments (see
Example 5). Overall, the cells maintained pluripotency when
transferred from planar substrates consisting of mixed cellulose
esters onto micro-carriers.
[0193] Next, H1 cells on the micro-carriers were differentiated to
definitive endoderm, according to the methods described in Example
7. After 4 days of differentiation, the H1 cells were released from
the micro-carriers and underwent FACS analysis showing greater than
65% of the cells expressing CXCR4 (FIG. 48). The cells were
efficiently differentiated into definitive endoderm regardless of
the micro-carrier type. There appeared to be a lower number of
cells differentiating into definitive endoderm on the HILLEX.RTM.II
(Solohill, MI) micro-carriers. The ability of the cells to
differentiate proves the flexibility of the expansion system.
Additionally cells can be grown and differentiated directly on
membranes and micro-carriers eliminating any need for an animal
component matrix.
TABLE-US-00001 TABLE 1 Attachment of H9 cells to micro-carrier
beads in MEF-CM in static cultures. attachment Bead company surface
coating 0-5* ProNectin F SoloHill .TM.-polystyrene Recombinant 0
fibronectin Plastic SoloHill .TM.-polystyrene none 0 Plastic Plus
SoloHill .TM.-polystyrene Cationic 0 HillexII SoloHill
.TM.-polystyrene Cationic trimethyl 2 ammonium Collagen SoloHill
.TM.-polystyrene Porcine collagen 0 FACTIII SoloHill
.TM.-polystyrene Cationic porcine 0 collagen Glass SoloHill
.TM.-polystyrene High silica glass 0 Cytodex 1 GE-dextran 0 Cytodex
3 GE-dextran denatured collagen 0 *5 is most efficient cell
attachment
TABLE-US-00002 TABLE 2 Attachment of H1 and H9 cells to
micro-carrier beads in MEF-CM with 10 .mu.M Rho kinase inhibitor,
Y27632. attachment Bead company surface coating 0-5* ProNectin F
SoloHill .TM.-polystyrene Recombinant 1 fibronectin Plastic
SoloHill .TM.-polystyrene none 1 Plastic Plus SoloHill
.TM.-polystyrene Cationic 1 HillexII SoloHill .TM.-polystyrene
Cationic trimethyl 4 ammonium Collagen SoloHill .TM.-polystyrene
Porcine collagen 1 FACTIII SoloHill .TM.-polystyrene Cationic
porcine 1 collagen Glass SoloHill .TM.-polystyrene High silica
glass 1 Cytodex 1 GE-dextran 4 Cytodex 3 GE-dextran denatured
collagen 4 *5 is most efficient cell attachment
TABLE-US-00003 TABLE 3 The population doublings for H1 and H9 cells
grown 5 passages on Cytodex 1 .RTM., Cytodex 3 .RTM., or HILLEX
.RTM. II. Cell line-micro- Population carrier doubling Standard
Deviation H9-HII 27 hrs 4.1 H9-C3 32.4 hrs 12.8 H1-C1 20.3 hrs 3.7
H1-C3 25 hrs 12.8
[0194] Publications cited throughout this document are hereby
incorporated by reference in their entirety. Although the various
aspects of the invention have been illustrated above by reference
to examples and preferred embodiments, it will be appreciated that
the scope of the invention is defined not by the foregoing
description but by the following claims properly construed under
principles of patent law.
* * * * *