U.S. patent application number 11/794262 was filed with the patent office on 2009-04-23 for stem cells culture systems.
Invention is credited to Eyal Banin, Pavel Itsykson, Benjamin Reubinoff, Etti Ben Shushan, Shelly Tannenbaum.
Application Number | 20090104695 11/794262 |
Document ID | / |
Family ID | 36615324 |
Filed Date | 2009-04-23 |
United States Patent
Application |
20090104695 |
Kind Code |
A1 |
Shushan; Etti Ben ; et
al. |
April 23, 2009 |
Stem Cells Culture Systems
Abstract
The present invention concerns systems and methods for providing
human cell cultures. Specific embodiments of the invention relate
to cultures of feeder cells for use in stem cell technology, as
well as cultures, culture systems and methods for maintenance and
propagating of stem cells in an undifferentiated state as well as
for the development of somatic cells cultures from stem cells, the
somatic cell cultures being free of extraembryonic cells.
Inventors: |
Shushan; Etti Ben;
(Jerusalem, IL) ; Tannenbaum; Shelly; (Efrat,
IL) ; Itsykson; Pavel; (Ramat Gan, IL) ;
Banin; Eyal; (Jerusalem, IL) ; Reubinoff;
Benjamin; (Mevaseret Zion, IL) |
Correspondence
Address: |
THE NATH LAW GROUP
112 South West Street
Alexandria
VA
22314
US
|
Family ID: |
36615324 |
Appl. No.: |
11/794262 |
Filed: |
December 29, 2005 |
PCT Filed: |
December 29, 2005 |
PCT NO: |
PCT/IL05/01397 |
371 Date: |
May 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60639809 |
Dec 29, 2004 |
|
|
|
Current U.S.
Class: |
435/366 ;
435/374; 435/375; 435/404 |
Current CPC
Class: |
C12N 2502/13 20130101;
C12N 2500/38 20130101; C12N 2501/115 20130101; C12N 5/0621
20130101; C12N 2500/44 20130101; C12N 2500/98 20130101; C12N
2500/99 20130101; C12N 2500/32 20130101; C12N 5/0606 20130101; C12N
2501/16 20130101; C12N 2501/155 20130101; C12N 2500/90 20130101;
C12N 2506/02 20130101; C12N 2533/52 20130101 |
Class at
Publication: |
435/366 ;
435/404; 435/374; 435/375 |
International
Class: |
C12N 5/02 20060101
C12N005/02; C12N 5/08 20060101 C12N005/08 |
Claims
1. A cell culture comprising cells obtained from human umbilical
cord tissue, the cells obtained from human umbilical cord being
capable of maintaining stem cells (SC) in an undifferentiated state
when co-cultured therewith.
2. The cell culture of claim 1, wherein the cells obtained from
human umbilical cord are feeder cells.
3. The cell culture of claim 1, wherein the lls obtained from human
umbilical cord are essentially fibroblast cells.
4. A culture system for maintenance of stem cells (SC) in an
undifferentiated state, the culture system comprising feeder cells
expanded from human umbilical cord cells, human embryonic
fibroblast cells (HEF) and a combination of same.
5. The culture system of claim 4, wherein said SC are embryonic
SC.
6. The culture system of claim 4, wherein said SC are human SC.
7. The culture system of claim 4, wherein said feeder cells
comprise fibroblast cells.
8. The culture system of claim 4, for propagation of
undifferentiated SC.
9. The culture system of claim 4, being an animal free culture
system.
10. The culture system of claim 4, comprising a humanized serum
replacement substitute.
11. The culture system of claim 4, being essentially free of
antibacterial agents and/or reducing agents.
12. An undifferentiated pluripotent human embryonic SC culture
obtained by incubating a cluster of cells from inside a blastocyst
with a culture system according to claim 4.
13. A method for maintaining SC in an undifferentiated state, the
method comprising incubating said cells with a culture system
comprising feeder cells expanded from human umbilical cord cells,
human embryonic fibroblast cells (HEF) or a combination of
same.
14. The method of claim 13, wherein said SC are embryonic stem
cells.
15. The method of claim 13, wherein said SC are human stem
cells.
16. The method of claim 13, wherein said feeder cells comprise
fibroblast cells.
17. The method of claim 13, for propagating said SC in an
undifferentiated state.
18. The method of claim 13, comprising incubating said SC with a
humanized serum replacement substitute.
19. The method of claim 13, wherein said culture system is
essentially free of antibacterial agents and/or reducing
agents.
20. A culture system for inhibiting or preventing differentiation
of stem cells to extraembryonic cells, the culture system
comprising nicotinamide (NA) or a derivative of NA having an
inhibitory effect on differentiation of SC to extraembryonic cells
similar to that of NA.
21. The culture system of claim 20, where in said SC are embryonic
stem cells.
22. The culture system of claim 20, wherein said SC are human stem
cells.
23. The culture system of claim 20, capable of inducing
differentiation of SC into somatic cells.
24. The culture system of claim 23, for inducing differentiation of
said SC to neural precursor cells.
25. The culture system of claim 24, for inducing differentiation of
said SC to retinal pigmented epithelial (RPE) cells.
26. An undifferentiated human embryonic SC culture essentially free
of extraembryonic cells, obtained by incubating a cluster of cells
from inside a blastocyst with a culture system according to claim
20.
27. A cell culture essentially free of extraembryonic cells and
comprising somatic cells obtained by incubating stem cells with a
culture system according to claim 20.
28. The cell culture of claim 27, wherein said somatic cells are
neural precursor cells.
29. The cell culture of claim 27, wherein said somatic cells are
retinal pigmented epithelial (RPE) cells.
30. A method for inhibiting or preventing differentiation of SC to
extraembryonic cells, the method comprises incubating said SC in a
culture system comprising NA or a derivative of NA having an
inhibitory effect on differentiation of SC to extraembryonic cells
similar to that of NA.
31. The method of claim 30, wherein said SC are embryonic SC.
32. The method of claim 30, wherein said SC are human SC.
33. The method of claim 30, wherein in the presence of said NA or
said derivative of NA, said SC differentiate to somatic cells.
34. The method of claim 33, wherein said somatic cells are neural
precursor cells.
35. The method of claim 33, wherein said somatic cells are retinal
pigmented epithelial (RPE) cells.
36. The method of claim 30, comprising incubating said SC in a
culture system comprising between about 1 mM to about 20 mM NA or a
NA derivative having an inhibitory effect on differentiation of SC
to extraembryonic SC similar to that of NA.
37. The method of claim 30, comprising culturing said SC in a
suspension.
38. The method of claim 30, for obtaining survival of said SC in
said culture system for at least 12 weeks.
39. The method of claim 30, wherein said NA induces an increase in
number of cells within embryoid bodies (EB).
40. A serum free culture system for maintenance of SC in an
undifferentiated state, the serum free culture system comprising
animal reagent free basic medium, and humanized serum replacement
substitute.
41. The serum free culture system of claim 40, being a humanized
culture system.
42. The serum free culture system of claim 40, wherein said basic
medium is selected from Cellgro Stem Cell Growth Medium, KO DMEM,
Neurobasal.TM., or X-Vivo 10.
43. The serum free culture system of claim 40, wherein said
humanized serum replacement substituent is selected from TCH.TM.,
Nutridoma-CS, N2 or combination of same.
44. The serum free culture system of claim 40, wherein when said
basic medium is Neurobasal.TM., the system further comprises a N2
supplement or a modification of N2 supplement.
45. The serum free culture system of claim 40, wherein said SC are
embryonic SC.
46. The serum free culture system of claim 40, wherein said SC are
human SC.
47. The serum free culture system of claim 40, comprising human
derived feeder cells.
48. The serum free culture system of claim 47, wherein the feeder
cells comprise fibroblast cells.
49. The serum free culture systems of claim 48, wherein said
fibroblast cells are selected from human embryonic fibroblast cells
(HEF), umbilical cord derived fibroblast cells and foreskin derived
fibroblast cells.
50. An undifferentiated human embryonic SC culture obtained by
incubating a cluster of cells from inside a blastocyst with a serum
free culture system according to claim 40.
51. A method of maintaining SC in an undifferentiated state, the
method comprises incubating said cells with a culture system
comprising serum free culture system comprising serum free basic
medium and humanized serum replacement substitute.
52. The method of claim 51, wherein said serum free culture system
comprises a e basic medium selected from Cellgro Stem Cell Growth
Medium, KO DMEM, Neurobasal.TM., or X-Vivo 10.
53. The method of claim 51, wherein said humanized serum
replacement substituent is selected from TCH.TM., Nutridoma-CS N2
supplement or a modification of N2 supplement or combination of
same.
54. The method of claim 51, wherein when said basic medium is
Neurobasal.TM., the system further comprises a N2 supplement or a
modification of N2 supplement.
55. The method of claim 51, for maintaining embryonic SC in an
undifferentiated state.
56. The method of claim 51, for maintaining human SC.
57. The method of claim 51, comprising incubating said cells with
human derived feeder cells.
58. The method of claim 57, wherein the feeder cells comprise
fibroblast cells.
59. The method of claim 58, wherein said fibroblast cells are
selected from human embryonic fibroblast cells (HEF), umbilical
cord derived fibroblast cells and foreskin derived fibroblast
cells.
60. A culture system for maintenance of a SC in an undifferentiated
state, the culture system comprising Neurobasal.TM. medium.
61. The culture system of claim 60, comprising N2 supplement or a
modification of N2 supplement.
62. The culture system of claim 60, for propagation of SC in a
suspension or in flat colonies.
63. The culture system of claim 60, wherein said SC are human
SC.
64. The culture system of claim 60, wherein said SC are embryonic
SC.
65. The culture system of claim 60, comprising one or more growth
factors for promoting growth of said undifferentiated hESC in
culture.
66. The culture system of claim 60, comprising an extracellular
matrix (ECM).
67. A cell culture of undifferentiated human embryonic SC obtained
by incubating a cluster of cells from inside a blastocyst with a
culture system according to claim 60.
68. The cell culture of claim 67, wherein said cells are maintained
in a suspension or as flat colonies.
69. A method for maintaining SC in an undifferentiated state, the
method comprising incubating said cells with a culture system
comprising Neurobasal.TM. medium.
70. The method of claim 69, comprising supplementing said medium
with N2 supplement or a modification of N2 supplement.
71. The method of claim 69, wherein said cells propagate in a
suspension or as flat colonies.
72. A method of obtaining a cell culture comprising cells obtained
from human umbilical cord tissue, the human umbilical cord derived
cells being capable of maintaining stem cells (SC) in an
undifferentiated state when co-cultured therewith, the method
comprises: (a) isolating umbilical cord cells from umbilical cord
tissue; (b) culturing said umbilical cord cells in a culture medium
including serum.
73. The method of claim 72, wherein said umbilical cord tissue is
obtained from healthy pregnant women undergoing elective Cesarean
sections at term.
74. The method of claim 72, wherein said umbilical cord cells are
obtainable by mincing said umbilical cord tissue into small
pieces.
75. The method of claim 74, wherein the minced umbilical cord
tissue pieces are fixed to walls of a container in the presence of
culture medium, and allowed to incubate undisturbed for a number of
weeks until the fibroblast cells begin to migrate out of the
umbilical cord pieces.
76. The method of claim 72, wherein said serum is human serum.
77. The method of claim 72, wherein said serum and/or said serum
replacement are provided at a concentration of at least 10%.
78. The method of claim 77, wherein said serum and/or said serum
replacement are provided at a concentration of 20%.
Description
FIELD OF THE INVENTION
[0001] The invention relates to stem cells (SC) in particularly to
methods and systems for handling human embryonic stem cells
(hESC).
LIST OF PRIOR ART
[0002] The following is a list of prior art, which is considered to
be pertinent for describing the state of the art in the field of
the invention. [0003] (1) Thomson, J. A. et al. Embryonic stem cell
lines derived from human blastocysts. Science 282, 1145-1147
(1998). [0004] (2) Reubinoff, B. E., Pera, M. F., Fong, C. Y.,
Trounson, A. & Bongso, A. Embryonic stem cell lines from human
blastocysts: somatic differentiation in vitro. Nat Biotechnol 18,
399-404 (2000) [0005] (3) Amit, M. et. al. Clonally derived human
embryonic stem cell lines maintain pluripotency and proliferative
potential for prolonged periods of culture. Dev Biol 227, 271-278
(2000). [0006] (4) Xu, C. et al. Feeder-free growth of
undifferentiated human embryonic stem cells. Nat Biotechnol 19,
971-974 (2001). [0007] (5) Amit, M. et al. Human feeder layers for
human embryonic stem cells. Biol Reprod 68, 2150-2156 (2003).
[0008] (6) Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C.
& Bongso, A. Human feeders support prolonged undifferentiated
growth of human inner cell masses and embryonic stem cells. Nat
Biotechnol 20, 933-936 (2002). [0009] (7) Cowan, C. A. et al.
Derivation of embryonic stem-cell lines from human blastocysts. N
Engl J Med 350, 1353-1356 (2004). [0010] (8) Amit, M., Shariki, C.,
Margulets, V. & Itskovitz-Eldor, J. Feeder layer- and
Serum-Free Culture of Human Embryonic Stem Cells. Biol Reprod
70(3):837-45 (2004). [0011] (9) Pera, M. F. et al. Regulation of
human embryonic stem cell differentiation by BMP-2 and its
antagonist noggin. J Cell Sci 117, 1269-1280 (2004). [0012] (10)
GB2409208. [0013] (11) WO 04/031343 [0014] (12) Xu, R. H., et al.
Basic FGF and suppression of BMP signaling sustain undifferentiated
proliferation of human ES cells. Nat. Methods. 3, 164-5 (2005)
[0015] (13) Vallier L, et al. Activin/Nodal and FGF pathways
cooperate to maintain pluripotency of human embryonic stem cells. J
Cell Sci. 118, 4495-509 (2005)
BACKGROUND OF THE INVENTION
[0016] Stem cells are immature, unspecialized cells that renew
themselves for long periods through cell division. Under certain
conditions, they can differentiate into mature, functional cells.
Human embryonic stem cells (hESC) are derived from early surplus
human blastocysts.sup.1, 2, Human ES cells are unique stem cells
since they can self-renew infinitely in culture, and since they
have a remarkable potential to develop into extraembryonic lineages
as well as all somatic cells and tissues of the human body.sup.1,
2.
[0017] Given the unique properties of hESC, they are expected to
have far-reaching applications in the areas of basic scientific
research, pharmacology, and regenerative medicine. Human ES cell
lines can provide a powerful in vitro model for the study of the
molecular and cellular biology of early human development, for
functional genomics, drug screening, and discovery. They may serve
for toxicology and teratogenicity high throughput screening. Since
hESC can self-renew indefinitely and can differentiate into any
cell type, they can serve as a renewable, unlimited donor source of
functionally mature differentiated cells or tissues for
transplantation therapy. In addition, transplanted
genetically-modified hESC can serve as vectors to carry and express
genes in target organs in the course of gene therapy.
[0018] While the promise of hESC for basic scientific research
pharmacology and regenerative medicine is remarkable, the
exploitation of hESC for most applications depends upon further
development. Improved control of the growth of undifferentiated
hESC, the development of bulk feeder-free cultures of
undifferentiated cells, the development of animal-free culture
systems, and the development of methods and tools which direct the
differentiation and generate pure cultures of mature functional
cells of a specific type are required.
[0019] At present, few culture systems are most commonly used to
propagate undifferentiated hESC.sup.1-4. In the initial culture
system that was developed, undifferentiated hESC are cultured in
serum-containing medium as colonies, upon a layer of fibroblast
feeder cells (of mouse.sup.1, 2 or human origin.sup.5, 11). It is
possible to remove all animal products from this culture system and
replace them with those from a human source.sup.6. It was found
that in this system the cells are propagated as clumps on a low
scale which does not allow cloning.sup.2.
[0020] An alternative culture system that was developed and used
extensively is a serum-free system that includes the knockout (KO)
medium supplemented with knockout serum replacement (KOSR) and
FGF2. This system allows cloning of undifferentiated hESC, although
at a low efficiency.sup.3. Undifferentiated cells are cultured as
flat colonies and may be propagated as small clusters or single
cells (by using trypsin.sup.7).
[0021] Another alternative culture system for use in the
proliferation of undifferentiated growth of hESC comprises a
culture matrix comprising extracellular matrix (ECM) prepared from
feeder cells and a conditioned medium being preconditioned by
feeder cells. The suggested leading cells in the feeder cells
include primary mouse embryonic fibroblasts (PMEF) a mouse
embryonic fibroblast cell line (MEF) murine foetal fibroblasts
(MFF) human embryonic fibroblasts (HEF) human foetal muscle (HFM)
human foetal skin cells (HFS) human adult skin cells, human
foreskin fibroblasts (HFF).sup.10 human adult Fallopian tubal
epithelial cells (HAFT) or human marrow stromal cells (HMSC).
[0022] Undifferentiated propagation may be accomplished with the KO
serum-free culture system without the use of feeders by plating and
growing colonies on extracellular matrices (ECM) within a
feeder-conditioned KO medium supplemented with KOSR and FGF2.sup.4.
Furthermore, it has been suggested that feeder conditioning may be
replaced by substituting the medium with high concentrations of
FGF2 and noggin.sup.12. Alternatively, feeder conditioning was
replaced by transforming growth factor .beta.1 and human LIF (in
addition to FGF2) and growing the cells on human fibronectin.sup.8.
In a recent publication, undifferentiated propagation of hESC
colonies, in the absence of feeders' was reported with a chemically
defined medium without serum replacer, supplemented with activin or
nodal plus FGF2.sup.13.
[0023] A key limitation of hESC culture systems is that they do not
allow the propagation of pure populations of undifferentiated stem
cells and their use always involves some level of background
differentiation. The stem cells most commonly follow a default
pathway of differentiation into an epithelial cell type that grows
either as a monolayer of flat squamous cells or form cystic
structures. Most probably, this form of differentiation represents
differentiation of hESC into extraembryonic endoderm.sup.9.
[0024] Spontaneous differentiation of hESC into presumably
extraembryonic lineages also interferes with the derivation of
somatic differentiated cells. Under various
differentiation-inducing conditions, such as in embryoid bodies
(EB) suspension cultures, differentiation into cystic
extraembryonic structures may be common or may predominate and
limit differentiation into somatic lineages. Control and
elimination of the differentiation into extraembryonic lineages
therefore, may be invaluable in the derivation of somatic lineages,
in addition to its importance in maintaining the stem cells in an
undifferentiated state. It has been recently demonstrated that
under differentiation-inducing culture conditions, the bone
morphogenetic protein (BMP) antagonist noggin can prevent
extraembryonic differentiation of hESC and promote their
differentiation into the neural lineage.sup.9.
SUMMARY OF THE INVENTION
[0025] In accordance with a first aspect, the present invention
provides a cell culture comprising cells obtained from human
umbilical cord tissue, the human umbilical cord derived cells being
capable of maintaining stem cells (SC) in an undifferentiated state
when co-cultured therewith. The human umbilical cord derived cells
are preferably used as feeder cells in SC cultures.
[0026] The invention also provides a first culture system for
maintenance of SC in an undifferentiated state, the culture system
comprising feeder cells expanded from human umbilical cord cells,
human embryonic fibroblast cells (HEF) and a combination of same.
According to one preferred embodiment, the culture system comprises
the human umbilical cord derived feeder cells of the invention.
[0027] Within this aspect of the invention there is also provided
an undifferentiated pluripotent human embryonic SC culture obtained
by incubating a cluster of cells from inside a blastocyst with the
first culture system of the invention.
[0028] The invention also provides a method for maintaining SC in
an undifferentiated state, the method comprising incubating said
cells with a culture system comprising feeder cells expanded from
human umbilical cord cells, human embryonic fibroblast cells (HEF)
or a combination of same.
[0029] The use of feeder cells expanded from human umbilical cord
derived cells, human embryonic fibroblast cells (HEF) and a
combination of same for the preparation of a culture system for
maintenance of SC in an undifferentiated state also forms part of
the invention.
[0030] In accordance with a second aspect, the invention provides a
further, second, culture system for inhibiting or preventing
differentiation of SC to extraembryonic cells, the culture system
comprising nicotinamide (NA) or a derivative of NA having an
inhibitory effect on differentiation of stem cells to
extraembryonic cells similar to that of NA.
[0031] A human embryonic SC culture essentially free of
extraembryonic cells is also provided in the context of this aspect
of the invention, the SC culture being obtained by incubating a
cluster of cells from inside a blastocyst with a culture system
comprising said NA or derivative thereof.
[0032] In accordance with this second aspect, there is also
provided a method for inhibiting or preventing differentiation of
stem cells to extraembryonic cells, the method comprises incubating
said stem cells in a culture system comprising NA or a derivative
of NA having an inhibitory effect on differentiation of stem cells
to extraembryonic cells similar to that of NA.
[0033] Further in accordance with this aspect of the invention
there is provided the use of NA or a NA derivative having an
inhibitory effect on differentiation of to extraembryonic cells
similar to that of NA for the preparation of a culture system for
inhibiting or preventing differentiation of SC to extraembryonic
cells.
[0034] In yet a third aspect of the invention there is provided a
further, third, culture system, a humanized culture system for
maintenance of SC in an undifferentiated state, the culture system
comprising an animal free basic stem cell culture medium and
humanized serum replacement substitute.
[0035] In accordance with this aspect there is also provided an
undifferentiated human embryonic SC culture obtained by incubating
a cluster of cells from inside a blastocyst with the humanized
culture system comprising the animal free stem cell basic culture
medium and a humanized serum replacement substitute.
[0036] In accordance with this aspect of the invention, there is
also provided a method of maintaining stem cells in an
undifferentiated state, the method comprises incubating said cells
with a culture system comprising animal free stem cell basic
culture medium and humanized serum replacement substitute.
[0037] In accordance with a fourth aspect of the invention there is
provided a culture system for maintenance SC in an undifferentiated
state, the culture system comprising Neurobasal.TM. medium.
[0038] Within this aspect there is also provided a culture of SC in
an undifferentiated state, the SC culture being obtained by
culturing a cluster of cells from inside a blastocyst with a
culture system comprising Neurobasal.TM. medium.
[0039] In accordance with this aspect of the invention there is
also provided a method for maintaining a culture of SC in an
undifferentiated state, the method comprising incubating said cells
with a culture system comprising Neurobasal.TM. medium as well as
the use of Neurobasal.TM. medium for the preparation of a culture
system for maintaining a suspension of stem cells in an
undifferentiated state.
[0040] The SC may be maintained in the Neurobasal.TM.-based culture
system in the form of a suspension as well as in a monolayer (flat
colonies). Preferably, the Neurobasal.TM. medium is supplemented
with N2 supplement or an N2 like supplement as defined below.
[0041] Finally, there is provided in accordance with the invention
a method of maintaining SC in an undifferentiated state comprising
culturing SC with feeder cells expanded from human umbilical cord
cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] In order to understand the invention and to see how it may
be carried out in practice, a preferred embodiment will now be
described, by way of non-limiting example only, with reference to
the accompanying drawings, in which:
[0043] FIGS. 1A-1D--are phase contrast images of cord fibroblasts
primary culture (FIG. 1A), human embryonic fibroblasts (FIG. 1B),
fibroblasts derived from umbilical cord (FIG. 1C) and from foreskin
(FIG. 1D).
[0044] FIG. 2A-2F--are immunofluorescent images of umbilical cord
foreskin, and human embryonic fibroblasts stained by anti-vimentin
antibody (FIGS. 2A, 2C and 2E, respectively) and the corresponding
DAPI nuclear counter staining (FIGS. 2B, 2D and 2F) showing that
the human feeders express vimentin.
[0045] FIG. 3--is a bar graph showing FACS analysis of the
percentage of feeders expressing CD44 and that are immunoreactive
with anti-fibroblast antibody indicating that a high percentage of
the feeders that are derived from the three sources express CD44
and are immunoreactive with anti-fibroblast antibody.
[0046] FIGS. 4A-4B--are representative analysis of one metaphase
plate of human embryonic fibroblasts (FIG. 4A) and foreskin (FIG.
4B) showing that the human feeders have a normal karyotype.
[0047] FIG. 5A-5C--are phase contrast images of colonies of
undifferentiated hESC that are cultured on three types of human
feeders, on umbilical cord derived feeders (FIG. 5A), human
embryonic fibroblasts (FIG. 5B) and on foreskin derived feeders
(FIG. 5C)
[0048] FIG. 6A-6L are representative FACS histograms of marker
expression by hESC cultured on the three feeder fibroblast types,
including expression of SSEA4, TRA1-60, TRA1-81 and SSEA1 by hESC
on cord derived feeders (FIGS. 6A-6D, respectively), by hESC on
human embryonic fibroblasts (FIG. 6E-6H, respectively) or by hESC
on foreskin (FIG. 6I-6L, respectively).
[0049] FIG. 7A-7C--are immunofluorescent images of hES colonies
expressing AP, when cultured on foreskin derived feeder cells (FIG.
7A), on umbilical cord derived feeder cells (FIG. 7B) and on human
embryonic fibroblast cells (FIG. 7C).
[0050] FIG. 8A-8F--are immunofluorescent images (FIGS. 8A, 8B and
8C) and the corresponding DAPI nuclear counter staining (FIGS. 8D,
8E and 8F) of hESC colonies expressing Oct4 when cultured on human
embryonic fibroblast cells (FIGS. 8A and 8D, cultured for 6 weeks),
on foreskin derived feeders (FIGS. 8B and 8E, cultured for 1 week)
and on umbilical cord derived feeder cells (FIGS. 8C and 8F,
cultured for 10 weeks).
[0051] FIG. 9--is a bar graph representing FACS analysis of the
percentage of hESC cultured on two independent cord derived feeder
cell lines (CORD1 and CORD2), and expressing the indicated markers
of undifferentiated pluripotent stem cells at early (1-5) and late
(6-10) passage levels, showing that the percentage of hESC
expressing these markers is stable during propagation, as
determined after 5, 8, 4 and 9 weeks of culture (5 W, 8 W, 4 W and
9 W).
[0052] FIG. 10--is a bar graph representing FACS analysis of the
percentage of hESC cultured on two independent foreskin-derived
feeder cell lines (OR2 and OR4), and expressing the indicated
markers of undifferentiated pluripotent stem cells at early (1-5)
and late (6-10) passage levels, showing that the percentage of hESC
expressing these markers is stable during propagation as determined
after 3, 6, and 8 weeks of culture (3 W, 6 W and 8 W).
[0053] FIG. 11--is a bar graph representing FACS analysis of the
percentage of hESC cultured on two independent human embryonic
fibroblast feeder cell lines (HEF1 and HEF2), and expressing the
indicated markers of undifferentiated pluripotent stem cells at
early (1-5) and late (6-10) passage levels, showing that the
percentage of hESC expressing these markers is stable during
propagation, as determined after 2, 5 and 10 weeks (2 W, 5 W and 10
W).
[0054] FIG. 12--is a bar graph representing analysis of the
percentage of hESC cultured on two independent cord derived feeder
cell lines (CORD1 and CORD2), and expressing Oct 4 at early (1-5)
and late (6-10) passage levels and which was found to be stable
during propagation as determined after 2, 3, 7 and 10 weeks (2 W, 3
W, 7 W and 10 W).
[0055] FIG. 13--is a bar graph representing analysis of the
percentage of hESC cultured on two independent foreskin-derived
feeder cell lines (OR2 and OR4), and expressing Oct 4 at early
(1-5) and late. (6-10) passage levels and which was found to be
stable during propagation as determined after 1, 2, 5 and 9 weeks
(1 W, 2 W, 5 W and 9 W).
[0056] FIG. 14--is a bar graph representing analysis of the
percentage of hESC cultured on two independent human embryonic
fibroblast cell lines (HEFG1 and HEFG2), and expressing Oct 4 at
early (1-5) and late (6-10) passage levels and which was found to
be stable during propagation as determined after 1 and 6 weeks (1 W
and 6 W).
[0057] FIGS. 15A-15I are immunofluorescent images of EBs-derived
differentiated cells expressing .beta.-tubulin (FIGS. 15A, 15D and
15G), AFP (FIGS. 15B, 15E and 15E), desmin (FIGS. 15C and 15I), or
muscle-actin (m-actin, FIG. 15F) when cultured on cord-derived
feeders (FIGS. 15A-15C); on human embryonic fibroblasts (FIGS.
15D-15F); and on foreskin derived feeders (FIGS. 15G-15I).
[0058] FIG. 16--is a bar graph showing the effect of bFGF at the
indicated concentration on the number of cells that were harvested
per flask at the time of the culture split as shown.
[0059] FIG. 17A-17D--are phase contrast images of cord-derived
feeders, showing the effect of bFGF on their morphology after
prolonged propagation in the presence of serum without
bFGF-supplementation (FIG. 17A) or with the two indicated
concentrations of bFGF supplementations (FIGS. 17B and FIG. 17C),
FACS analysis of the percentage of feeders expressing CD44 and that
are immunoreactive with anti-fibroblast antibody (Anti-fib ab) is
also shown (FIG. 17D). Analysis was performed at passage 10 in the
presence of serum, and at passage 17 when the medium was
supplemented with 5 ng/ml and 10 ng/ml of bFGF.
[0060] FIGS. 18A-18F--are phase contrast images (FIGS. 18A-18C) and
immunofluorescent images (FIGS. 18D-18F) of hESC colonies cultured
on cord-derived fibroblasts that were propagated for 17 passages in
the presence (FIGS. 18B, 18C, 18E and 18F) or absence (FIGS. 18A
and 18D) of bFGF. The cord-derived fibroblasts supported
undifferentiated proliferation of the hESCs as determined by the
expression of alkaline phosphatase by the hESC (FIGS. 18D-18F) and
the expression of stem cell markers by a high percentage of the
hESCs (FACS analysis, FIG. 18G).
[0061] FIG. 19--is a phase contrast micrograph of hESC cultured on
HEF feeder layer in Cellgro medium supplemented with 1% TCH showing
that hESC retain the morphology of undifferentiated pluripotent
stem cells when TCH is used as the serum replacement
supplement.
[0062] FIG. 20--is a bar graph showing the percentage of SSEA-4
expressing on hESC, when cultured on a foreskin or HEF feeder
layers, being similar when the KO DMEM was supplemented with KO SR,
2% TCH, or 2% Nutridoma and showing that Nutridoma-CS is as
effective as TCH in supporting undifferentiated propagation of
hESC.
[0063] FIG. 21--is a phase contrast micrograph of hESC colonies
cultured within NBN2 showing that hESCS retain the morphology of
undifferentiated cells when colonies are cultivated on human
feeders in NBN2.
[0064] FIG. 22A-22D--are dark field micrographs of small
transparent clusters of cells that develop 7 days after transfer of
undifferentiated hESCs into suspension culture within NBN2 medium
(FIG. 22A) and after 3 weeks in suspension culture within NBN2,
indirect immunofluorescent analysis showed that the majority of
hESCs express SSEA4 (FIG. 22B) and Oct4 (FIG. 22D). Nuclei of cells
in D are counterstained with DAPI in (FIG. 22C).
[0065] FIG. 23A-23B--are bar graphs showing the percentage of
SSEA-4+ cells (FIG. 23A) and total number of cell/well (FIG. 23B)
as analyzed after 3 weeks suspension culture of equal initial
numbers of hESC in NBN2 medium+FGF2 supplemented with various
combinations of ECM components and factors.
[0066] FIG. 24A-24D--are dark field micrographs of EBs that were
cultured for 4 weeks in the presence and absence of NA (the culture
medium included 10% FCS). EBs with typical cystic structures
(cystic EBs) developed in the absence of NA (FIGS. 24A and 24B),
while in the presence of NA, cystic formation was not observed and
the EBs were comprised of tightly packed cells (FIGS. 24C and
24D).
[0067] FIGS. 25A-25P--are dark field micrographs of EBs that were
cultured for 2-5 weeks in chemically-defined medium (NBN2) in the
presence or absence of NA and retinoic acid as indicated. EBs with
typical cystic structures (cystic EBs) developed in the absence of
NA (FIGS. 25A-25D, i.e. upper panel). In the presence of NA, cystic
formation was not observed, the EBs were comprised of tightly
packed cells and were significantly larger (FIGS. 25E-25H, i.e.
second panel). In the presence of RA, the EBs were smaller and
included multiple cysts (FIGS. 251-25L, i.e. third panel). NA
blocked the effects of RA (FIGS. 25M-25P, i.e. lower panel).
[0068] FIG. 26--is a RT-PCR analysis demonstrating that the
expression of the endodermal marker .alpha.-fetoprotein is
suppressed within EBs that differentiated in the presence of NA in
comparison to control EBs that were cultured in the absence of NA.
After 4 weeks of differentiation, the effect of NA was more
prominent in comparison to the effect after 2 weeks.
[0069] FIGS. 27A-27D--are immunocytochemical studies demonstrating
suppressed expression of .alpha.-fetoprotein (AFP) and
cytokeratin-8 (CK) in EBs that differentiated in the presence of
NA. Following 4 weeks of differentiation in the presence of NA,
only a few cells in sections of EBs were immunoreactive with
anti-.alpha.-fetoprotein (FIG. 27A) and cytokeratin-8 (FIG. 27B).
Cells that expressed .alpha.-fetoprotein (FIG. 27C) and
cytokeratin-8 (FIG. 27D) were abundant within sections of control
EBs that differentiated in the absence of NA.
[0070] FIG. 28A-28D--are dark field micrographs of EBs
differentiating in the presence of NA showing that the percentage
of EBs that included clusters of differentiated cells expressing
melanin increased with time (FIGS. 28A, 28B, 28C and 28D
representing results after 2, 4, 6 and 12, respectively).
[0071] FIG. 29--is a real time PCR analysis of EBs differentiated
for 6 weeks in the presence or absence of NA, demonstrating the
induction of expression of RPE markers by NA.
[0072] FIGS. 30A-30H--are images showing that melanin-expressing
cells that were generated in the presence of NA had morphological
characteristics that are typical of RPE cells. Specifically shown
are dark field micrograph (FIG. 30A), phase contrast image (FIG.
30B) and indirect immunofluorescent stainings of RPE cells markers
including ZO-1 (FIG. 30C), Pax6 (FIG. 30D), MITF (FIG. 30E), CRALBP
(FIG. 30F), Bestrophin (FIG. 30G) and RPE65 (FIG. 3011).
[0073] FIG. 31--is RT-PCR analysis showing the expression of
chordin-like 1 by cells within LBs that were developed in the
presence of NA.
[0074] FIG. 32A-C--are H&E and fluorescent images demonstrating
the survival of transplanted hESC-derived RPE cells and their
integration within the host RPE layer of cells. An H&E image
showing the survival of an intra-vitreal graft, 4 weeks after
transplantation into the eye of a mature RCS rat (FIG. 32A). The
graft includes melanin expressing cells (dark pigmented cells).
Indirect immunofluorescent staining demonstrates that the cells
within the graft express GFP (white spots), confining their human
identity (FIG. 32B). Integration of transplanted hESC-derived RPE
cells (pigmented cells marked with arrows) in the albino rat RPE
layer is also demonstrated (FIG. 32C). Pigmented cells were not
observed in the RPE layer of control non transplanted eyes.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0075] The invention is described in the following detailed
description with reference to cell cultures and culture systems for
handling stem cells, preferably human embryonic stem cells. It
should be noted that in addition to the cell cultures and culture
systems discussed in detailed hereinbelow, also encompassed within
the present invention are uses of specific components described
with reference to the culture system in the preparation of such
culture systems as well as to methods of use of the culture system
in handling stem cells cultures and methods of preparing culture
cells.
[0076] As used in the specification and claims, the forms "a", "an"
and "the" is include singular as well as plural references unless
the context clearly dictates otherwise. For example, the term "a
culture system" includes one or more culture systems.
[0077] As used herein, the term "or" means one or a combination of
two or more of the listed choices
[0078] Further, as used herein, the term "comprising" is intended
to mean that the methods or composition includes the recited
elements, but not excluding others. Similarly, "consisting
essentially of" is used to define methods and systems that include
the recited elements but exclude other elements that may have an
essential significance on the functionality of the culture systems
of the inventions. For example, a culture system consisting
essentially of a basic medium, medium supplements and feeder cells
will not include or include only insignificant amounts (amounts
that will have an insignificant effect on the propagation and
differentiation of cells in the culture system) of other substances
that have an effect on cells in a culture. Also, a composition
consisting essentially of the elements as defined herein would not
exclude trace contaminants from the isolation and purification
method. "Consisting of" shall mean excluding more than trace
elements of other elements. Embodiments defined by each of these
transition terms are within the scope of this invention.
[0079] Further, all numerical values, e.g., concentration or dose
or ranges thereof, are approximations which are varied (+) or (-)
by up to 20%, at times by up to 10% of from the stated values. It
is to be understood, even if not always explicitly stated that all
numerical designations are preceded by the term "about". It also is
to be understood, although not always explicitly stated, that the
reagents described herein are merely exemplary and that equivalents
of such are known in the art.
[0080] In its broadest sense, the present invention concerns
culture cells, systems and methods for use of same in culturing of
stein cells. As used herein, the term "stem cells" refers to cells
which are capable of differentiating into other cell types having a
particular, specialized function (i.e., "fully differentiated"
cells) or self renewing and remaining in an undifferentiated
pluripotential state as detailed below.
[0081] As used herein, the term "cell" refers to a single cell as
well as to a population of (i.e. more than one) cells. The
population may be a pure population comprising one cell type.
Alternatively, the population may comprise more than one cell type.
Furthermore, as used herein, the term "cell culture" refers to any
in vitro culture of cells. Included within this term are continuous
cell lines (e.g. with an immortal phenotype), primary cell
cultures, finite cell lines (e.g., non-transformed cells), and any
other cell population maintained in vitro.
[0082] As used herein, the term "primary cell" is a cell which is
directly obtained from a tissue or organ of an animal, including a
human, in the absence of culture. Typically, though not
necessarily, a primary cell is capable of undergoing ten or fewer
passages in vitro before senescence and/or cessation of
proliferation. In contrast, a "cultured cell" is a cell which has
been maintained and/or propagated in vitro for ten or more
passages
[0083] Non-limiting examples of stem cells are hematopoietic stem
cells obtained from bone marrow tissue of an individual at any age
or from cord blood of a newborn individual, embryonic stem (ES)
cells obtained from the embryonic tissue formed after gestation
(e.g., blastocyst), or embryonic germ (EG) cells obtained from the
genital tissue of a fetus any time during gestation, preferably
before 10 weeks of gestation. Preferred stem cells according to the
present invention are human stem cells, more preferably, hESC.
[0084] Stem cells can be obtained using well-known cell-culture
methods. For example, hESC can be isolated from human blastocysts.
Human blastocysts are typically obtained from human in vivo
preimplantation embryos or from in vitro fertilized (IVF) embryos.
Alternatively, a single cell human embryo can be expanded to the
blastocyst stage. For the isolation of human ES cells the zona
pellucida is removed from the blastocyst and the inner cell mass
(ICM) is isolated by immunosurgery, in which the trophectoderm
cells are lysed and removed from the intact ICM by gentle
pipetting. The ICM is then plated in a tissue culture flask
containing the appropriate medium which enables its outgrowth.
Following 9 to 15 days, the ICM derived outgrowth is dissociated
into clumps either by a mechanical dissociation or by an enzymatic
degradation and the cells are then re-plated on a fresh tissue
culture medium. Colonies demonstrating undifferentiated morphology
are individually selected by micropipette, mechanically dissociated
into clumps, and re-plated. Resulting ES cells are then routinely
split every 1-2 weeks. For further details on methods of
preparation human ES cells see Thomson et al., [U.S. Pat. No.
5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133,
1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; as well as Bongso
et al., [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil.
Steril. 69:84, 1998].
[0085] Commercially available stem cells can be also be used in
accordance with the invention. Human ES cells can be purchased from
the NIH human embryonic stem cells registry. Non-limiting examples
of commercially available embryonic stem cell lines are BGO1, BGO2,
BGO3, BGO4, CY12, CY30, CY92, CY10, TE03 and TE32.
[0086] Potential applications of hESC are far ranging and include
drug discovery and testing, generation of cells, tissues and organs
for use in transplantation, production of biomolecules, testing the
toxicity and/or teratogenicity of compounds' and facilitating the
study of developmental and other biological processes. For example,
diseases presently expected to be treatable by therapeutic
transplantation of hESC or hESC derived cells include Parkinson's
disease, cardiac infarcts, juvenile-onset diabetes mellitus, and
leukemia [Gearhart J. Science 282: 1061-1062, 1998; Rossant and
Nagy, Nature Biotech. 17: 23-24, 1999].
[0087] There are, however, significant hurdles to the practical
exploitation of hESC. To maintain hESC in an undifferentiated
pluripotential state, the cells are usually cultured on feeder
cells. The feeder cells can secrete factors needed for stem cell
self-renewal and proliferation, while at the same time, inhibit
their differentiation.
[0088] Commonly used feeder cells includes a primary mouse
embryonic fibroblast (PMEF), a mouse embryonic fibroblast (MEF), a
murine fetal fibroblast (MFF), a human embryonic fibroblast (HEF),
a human fetal muscle cell (HFM), a human fetal skin cell (HFS), a
human adult skin cell, a human foreskin fibroblast (HFF), a human
adult fallopian tubal epithelial cell (HAFT) and a human marrow
stromal cells (hMSCs).
[0089] As used herein, the term "undifferentiated pluripotential
hES cells" or "hESC" refers to human precursor cells that have the
ability to form any adult cell. Such cells are true cell lines in
that they (i) are capable of indefinite proliferation in vitro in
an undifferentiated state; and (ii) are capable of differentiation
to derivatives of all three embryonic germ layers (endoderm,
mesoderm, and ectoderm) even after prolonged culture. Human ES
cells are derived from fertilized embryos that are less than one
week old.
[0090] Pluripotent SC present at their surface or express
biological markers which are used to identify pluripotent SC as
well as to verify that the cells in the culture are maintained in
an undifferentiated state [Thomson J A et al. Embryonic Stem Cell
Lines Derived from Human Blastocysts Science 282(5391):1145-1147
(1998)]. A non-limiting list of such cell markers comprise
stage-specific embryonic antigen such as SSEA-3, SSEA-4; antibodies
to specific extracellular matrix molecule which are synthesized by
undifferentiated pluripotent SC, such as TRA-1-60, TRA-1-81 and
GCTM-2; elevated expression of alkaline phosphatase which is
associated with undifferentiated pluripotent SC; transcription
factors unique to pluripotent SC and which are essential for
establishment and maintenance of undifferentiated SC, such as,
OCT-4 and Genesis [Carpenter, m. k., Rosier, E., Rao M. S.,
Characterization and Differentiation of Human Embryonic Stem Cells.
Cloning and Stem Cells 5, 79-88, 2003].
[0091] While widely used, human SC cultures based on murine derived
feeder cells, are less desired. Non-species specific feeder cell
technology reduces the value of stem cell cultures due to the
foreign nature of the source of the feeder cell. For example, such
non-species specific feeder cells contain both foreign cells and
foreign growth factors. Further, it is believed that the use of
non-species specific feeder cells in combination with different but
desirable cultured cells cannot provide the optimum growth
conditions as species specific derived feeder cells or conditioned
media. The issue of cross-species contamination is particularly
relevant to agricultural animals, endangered species, laboratory
animals, non-human primate cells, and hESC. It has been shown that
hESC are contaminated by foreign molecules when cultured with
mouse-derived feeders (Martin, M. J., et al., Human embryonic stem
cells express an immunogenic nonhuman sialic acid. Nat. Med. 2005;
11: 228-32). Contamination of hESC by mouse derived
molecules/pathogens may interfere with their exploitation as a
model for basic research and raises concerns as to their use in
transplantation therapy. Still further, non-human feeder cell
technology reduces the value of human derived SC cultures, as, for
example, such non-human feeder cells contain both non-human cells
and non-human growth factors. Also, it is believe that the use of
non-human feeder cells in combination with human cultured cells
cannot provide the optimum growth conditions as human derived
feeder cells.
[0092] Thus, the present invention provides, in accordance with a
first of its aspects, a cell culture derived from human umbilical
cord tissue, preferably excluding hematopoietic tissue, and being
capable of maintaining SC in an undifferentiated state when
co-cultured therewith. These feeder cells are obtained from
culturing, preferably in an animal free culture system, of cells
taken from umbilical cord tissue under conditions which allow the
cells to propagate/expand and isolating the thereby propagated
cells. The cells in the culture are essentially fibroblast cells
and are preferably used as feeders in stem cell culture
systems.
[0093] The cell cultures (either the feeder cells or the SC
cultures) in accordance with the invention may be a fresh cell
culture, cryopreserved culture as well as cryopreserved and thawed
cells.
[0094] As used herein, the term "derived" which may be used
interchangeably with the term "obtained" when used in the context
of cell formation denotes the development of a new cell line from
another cell line. For example, human embryonic cord derived cells
denote, in accordance with one embodiment of the invention,
fibroblast cells originating from embryonic cord tissue, which
under suitable condition propagate into a fibroblast cell line.
[0095] Further, as used herein, the term "feeder cells" which is
known interchangeably with "feeders" denotes any type of cells
which may be used as a substratum for other cells attachment and
growth in a culture system. Feeder cells are typically used to
allow growth and survival of single undifferentiated stem cells.
The Feeder cells provide conditions that maintain cell
proliferation, inhibit cell differentiation and preserve
pluripotency. Specifically, the feeder cells are cells that secrete
factors needed for stem cell proliferation, while inhibit their
differentiation. Methods of preparing feeder cells are well known
in the art (see, for example, U.S. patent Pub. No. 20030143736).
Generally, the feeder cells may be fibroblasts or other types of
cells, and the cells are inactivated by large-dose radiation before
use, such as .gamma.-ray, or by drugs, such as mitomycin C. After
the inactivation process, the surviving cells lost the capability
to proliferate, but retained their physiological functions, such as
metabolism and synthesis of growth factors.
[0096] As indicated above, the feeder cells are derived (expanded)
from umbilical cord tissue. Umbilical cord tissue may be obtained
in the course of vaginal delivery. However, a major advantage of
using umbilical cord tissue is that it may be obtained during
elective cesarean section in a sterile environment of an operating
theater. Moreover, the umbilical cord is obtained from the sterile
environment of the amniotic sac and has not been exposed to any
external contagious agents prior to donation. The sterile nature of
umbilical cord donation allows the derivation of feeders from the
umbilical cord tissue without the use of antibiotics or anti-fungal
drugs. Avoiding the use of anti-bacterial and anti-fungal drugs is
an advantage since these drugs may interfere with the growth of
cells in culture, alter the results of basic science studies and
most importantly may induce allergic reactions in recipients of
cells that were cultured in the presence of these drugs. Derivation
of feeders from other human primary tissues such as foreskin or
aborted fetuses are done under significant less sterile conditions.
The foreskin is exposed to bacteria that colonize the genital area
and it may be disinfected but not sterilized. Aborted fetuses are
also exposed to potential contamination by vaginal and genital
flora during dilatation and curettage.
[0097] An additional advantage of umbilical cord as opposed to
foreskin or human fetal tissues is that a significant volume of
blood may be sampled from the umbilical cord, tested for contagious
agents and archived. This is not possible with foreskin tissues
donated by newborn babies or with aborted fetuses. Lastly,
umbilical cord is routinely discarded and its donation is not
associated with emotional or moral constrains, while donation of
fetal tissues raises ethical concerns and is not morally accepted
by many.
[0098] In this connection there is thus also provided a method for
preparing umbilical cord derived feeder cells, the method
comprising isolating umbilical cord cells from umbilical cord
tissue and culturing said umbilical cord cells in a culture medium
including serum, thereby preparing said human umbilical cord feeder
cells. The umbilical cord cells may be isolated from the umbilical
cord tissue by mincing the tissue and affixing the umbilical cord
to a wall, such as a wall of a flask, and allowing the cells to
incubate undisturbed for a number of weeks until fibroblast cells
begin to migrate out of the minced umbilical cord tissue.
[0099] The umbilical cord tissue may be obtained from healthy
pregnant women undergoing elective Cesarean sections at term.
[0100] In accordance with the invention there is also provided a
culture system for maintaining stem cells (SC) in an
undifferentiated state, the culture system comprising feeder cells
selected from cells obtained from human umbilical cord tissue
(excluding cells obtained from umbilical cord blood), human
embryonic fibroblast cells (HEF) or a combination of same. The
culture system according to this aspect of the invention is term
herein the "human derived feeder cell aspect of the invention".
[0101] As used herein with respect to all aspects of the invention,
the terms "maintenance" means continued survival of a cell or
population of cells, at times, with an increase in numbers of
cells. "Proliferation", "propagation", "expansion" and "growth",
which are used interchangeably, refer to such an increase in cell
number. According to one embodiment, when referring to maintenance
of hESC on feeder cells, this term refers to a continuous survival
of the cells for at least 10 weeks.
[0102] The culture systems in accordance with the invention are
preferably for enabling maintenance of a population of stem cells
when cultured on feeder cells, and at time, propagation of same,
for a prolonged period of time, the period of time being at least
10 weeks.
[0103] When the feeder cells are derived from human umbilical cord
tissue, the feeder cells are essentially fibroblast cells. The term
"essentially fibroblast cells" denotes that the feeder cells
comprise in its majority fibroblasts, i.e. at least 70 of the cells
in the feeder cell population are fibroblast, preferably 85%, a
most preferably all the cells, i.e. essentially 100% of the feeder
cells are fibroblasts.
[0104] In accordance with one embodiment, the feeder cells are
provided in a form of a monolayer coated culture dish to which a
nutrient medium is added along with the culture cells. As used
herein, the terms "monolayer", "monolayer culture" and "monolayer
cell culture" refer to cells that have adhered to a substrate and
grow as a layer that is one cell in thickness. Monolayer cells may
be grown in any format, including but not limited to flasks, tubes,
coverslips (e.g., shell vials), roller bottles, etc. Monolayer
cells may also be grown attached to microcarriers, including but
not limited to beads. At times, the term monolayer also includes
growth of cells as flat colonies.
[0105] The term "culture system" denotes a combination of elements,
such as an extracellular matrix (ECM) and a culture (nutrient)
medium which together provide suitable conditions that support SC
growth. The conditions are such that SC can proceed through the
cell cycle, grow and divide. Preferably, the conditions are such
which enable growth of human stem cells, preferably, hESC. Further,
the culture system provides conditions that permit the embryonic
stem cells to stably proliferate in the culture system for at least
10 weeks. The nutrient medium may contain any of the following
appropriate combinations: a basic medium (a cell culture medium
usually comprising a defined base solution, which includes salts,
sugars and amino acids) as well as serum or serum replacement, and
other exogenously added factors. It is not intended that the term
"culture medium" or "nutrient medium" be limited to any particular
culture medium. For example, it is intended that the definition
encompass outgrowth as well as maintenance media. In accordance
with the human derived feeder cell aspect of the invention, the
culture system also comprises the feeder cells. However, the feeder
cells may be substituted with components derived from feeder cells
or other known and acceptable substitutes thereof, e.g. when
referring to other culture systems disclosed herein.
[0106] In accordance with one embodiment, the culture system is
employed for maintaining hESC in an undifferentiated pluripotential
state, as evidenced in the following non-limiting examples by the
expression of proteins such as SSEA-4, TRA-1-60, OCT-4, APase, but
not SSEA-1. Methods of preparing culture systems for culturing hESC
are well known in the art [see, for example, Reubinoff Be. et. al.,
Nat. Biotechnol. 18:399-404, 2002; Richards, M. et al., Nat.
Biotechnol. 20:933-936, 2002].
[0107] A hESC medium may typically contain 80% Dulbecco's Modified
Eagles Medium (DMEM), 20% defined Fetal Calf Serum, 1% L-Glutamine,
0.5% penicillin/streptomycin, 1% non-essential amino acids, 1%
Insulin-Transferrin-Selenium G supplement and 1 mM
.beta.-mercaptoethanol.
[0108] In an animal free culture system, which provides a
pathogen-free environment for the growth of ES cells, the cultures
rely on human feeder layers supplemented with human serum or serum
replacement suitable for the growth of human stem cells. The feeder
cells may be any suitable cells from human source as known in the
art or the isolated umbilical cord derived feeder cells of the
invention; the stem cells medium DMEM (used as the basic media) may
be replaced with KO DMEM (Gibco, or, equivalent), X-Vivo 10
(Biowhittaker, Maryland, or equivalent) or Cellgro Stem Cell Growth
Medium (CellGenix, Freiburg, Germany, or equivalent); the FCS may
be replaced with humanized serum replacement substitute, such as
TCH (Protide Pharmaceuticals, St. Paul, Minn., or equivalent) or
Nutridoma-CS (Roche, Germany, or equivalent). Since the animal free
system provides a pathogen free environment, reducing agents such
as .beta.-mercaptoethanol and antibacterial agents such as
penicillin/streptomycin) may be eliminated.
[0109] In the context of the human derived feeder cell culture
system aspect of the invention there is also provided a method for
maintaining stem cells in an undifferentiated state, the method
comprises incubating (co-culturing) said cells with a culture
system comprising feeder cells selected from human umbilical cord
tissue derived cells, human embryonic fibroblast cells (HEF) or a
combination of same.
[0110] According to one embodiment, the stem cells are incubated in
a culture system where the feeder cells are preferably provided as
a layer of cells, preferably a mono-layer, formed on a base of
culture dish. The culture system is then provided with a growth
environment, typically, an environment in which cells of interest
will proliferate in vitro. Temperatures of 37.degree. C. and 5%
CO.sub.2 in air are generally adopted.
[0111] In cultures of undifferentiated hESCs there is always some
level of background extraembryonic differentiation. Further, in
currently-used systems for the cultivation of undifferentiated
hESCs, or for induction of their differentiation towards somatic
lineages, three is tendency of hESCs to differentiate towards
extraembryonic lineages. In addition, upon induction of
differentiation, the default pathway of differentiation towards
extraembryonic lineages may predominate, and limit differentiation
into desired somatic lineages.
[0112] Thus, the invention also provides a culture system for
inhibiting or preventing differentiation of stem cells towards
extraembryonic lineages (to extraembryonic cells). The culture
system in accordance with this aspect of the invention comprise NA
(NA) or a derivative of NA having an inhibitory effect on
differentiation of stem cells towards extraembryonic lineages (to
extraembryonic cells) similar to that of NA. This aspect of the
invention is referred to herein as the "nicotinamide aspect of the
invention".
[0113] NA is a form of Vitamin B3 that may preserve and improve
beta cell function. NA is essential for growth and conversion of
foods to energy and it has been used in diabetes treatment and
prevention. It has now been found that NA is capable of inhibiting,
preferably, preventing differentiation of embryonic stem cells
towards extraembryonic lineages (to extraembryonic cells).
[0114] The term "derivative of nicotinamide" as used herein denotes
a compound which is a chemical modification of the natural NA.
##STR00001##
[0115] The chemical modification may include substitution on the
pyridine ring of the basic NA structure (via the carbon or nitrogen
member of the ring), via the nitrogen or the oxygen atoms of the
amide moiety, as well as deletion or replacement of a group, e.g.
to form a thiobenzamide analog of NA, all of which being as
appreciated by those versed in organic chemistry. The derivative in
the context of the invention also includes the nucleoside
derivative of NA (e.g. nicotinamide adenine). A variety of NA
derivatives are described, some also in connection with an
inhibitory activity of the PDE4 enzyme [WO03068233; WO02060875;
GB2327675A], or as VEGF-receptor tyrosine kinase inhibitors
[WO01/55114]. For example, the process of preparing
4-aryl-nicotinamide derivatives are described in WO05014549A. The
NA derivatives in the context of the invention are compound
determined to have an inhibitory effect, preferably preventative
effect, on differentiation of stem cells to extraembryonic lineages
(extraembryonic cells), similar to that of NA.
[0116] The effect of NA may be the result of inhibition of poly
(ADP-ribose) polymerase (PARP). Therefore the effect of NA may be
also achieved by treating the cells with other PARP inhibitors such
as 3-aminobenzmide, PJ-34 or 1, 5-dihydroxyisoquinoline. These
other PARP inhibitors are also included in the context of the term
"modification of NA". Yet further, the effect of NA may also be
attributed to the inhibition of SIRT protein deacetylase. Therefore
its effect may be also obtained by other SIRT inhibitors such as
splitomicin and sirtinol, which are thus, also included in the
context of the term term "modification of NA".
[0117] In accordance with the NA aspect of the invention, the stem
cells may be as described above, i.e. they may be stem cells from
any source, but are preferably human stem cells, further
preferably, human embryonic stem cells.
[0118] As used herein "inhibition of extraembryonic
differentiation" used synonymy with the term "prevention of
extraembryonic differentiation" denotes the maintenance as well as
the expansion of embryonic stem cell in a cell culture and that the
resulting cell culture is essentially free of extraembryonic cells
or membranes. The term "essentially free" is used to exclude
extraembryonic cells that may have an essential significance on the
functionality of the stem or somatic cells in the culture or that
the amount of the extraembryonic cells in the cell culture is
insignificant (an amount that will have an insignificant effect on
the propagation and differentiation of cells in the culture
system).
[0119] It is well appreciated that if extraembryonic
differentiation is essentially eliminated, a key challenge is to
further direct differentiation into a specific somatic lineage and
into a specific type of cell. It has now been found that
supplementation of a culture medium with NA can prevent the default
differentiation of hESCs towards extraembryonic lineages. It may
also direct the differentiation towards specific somatic lineage
such as but not limited to neural differentiation. The examples
provided herein show differentiation to neural precursor cells.
[0120] Proliferation and differentiation of embryonic stem cells
into insulin-producing cells in the presence of NA was suggested by
Vaca P et al, [Transplant Proc. 35(5):2021-3 2003] Specifically, it
was shown that while proliferation within EBs with or without
supplementation of the medium with NA is similar (FIG. 1A in Vaca
P. et al.), insulin content is increased in cells that
differentiate in the presence of NA. Nevertheless it is unclear
whether the increased insulin content was not related to increased
uptake of insulin from the medium.
[0121] It has now been found that NA effectively induced
differentiation of stem cells into somatic cells. Specifically,
albeit, not exclusively, NA was shown to induce differentiation to
neural cells, and within the neural lineage, NA treatment was found
to promote differentiation towards retinal pigmented epithelial
(RPE) cells. The use of RPE cells in transplantation has already
been described [Haruta, M. et al., In vitro and in vivo
characterization of pigment epithelial cells differentiated from
primate embryonic stem cells. Invest Ophthalmol V is Sci,
45:1020-1025 (2004)]. Thus, it is to be understood that the RPE
cells obtained in accordance with the present invention have
various therapeutic applications. One such application includes
transplantation of such cells in the eye to replenish
malfunctioning or degenerated RPE cells in retinal degenerations.
Genetically modified RPE cells may serve as a vector to carry and
express genes in the retina after transplantation. Other
applications may be the use of hESC-derived RPE cells as an in
vitro model for the development of new drugs to promote RPE
survival and function. hESC-derived RPE cells may serve for high
throughput screening for compounds that are toxic, trophic, induce
differentiation proliferation and survival of RPE cells. They may
be used to uncover mechanisms, new genes, soluble or membrane-bound
factors that are important for the development, differentiation,
maintenance, survival and function of photoreceptor cells.
[0122] The culture system in the NA aspect of the invention
comprises standard elements of culture media, as defined above
combined with NA. The concentration of NA in the medium may vary,
however, will preferably be in a concentration range between about
1 mM to about 20 mM, more preferably at a concentration of about 10
mM.
[0123] In the context of this aspect of the invention there is also
provided a method for inhibiting or preventing differentiation of
stem cells towards extraembryonic lineages (to extraembryonic
cells), the method comprises incubating said stem cells in a
culture system comprising NA or a derivative of NA as defined
above.
[0124] It should be noted that in the context of the present
invention the NA based culture systems was also effective for
increasing the survival of SC in the culture system. According to
one embodiment, cells survived in the culture for at least 12
weeks.
[0125] It should also be noted that the NA based culture system of
the invention was effective to induces an increase in number of
cells within embryoid bodies (EB) cultured therein.
[0126] For induction of somatic differentiation, the stem cells in
accordance with the NA aspect of the invention are preferably grown
as free floating clusters in a suspension. As used herein, the
terms "suspension" and "suspension culture" refer to cells that
survive and proliferate without being attached to a substrate.
[0127] A further aspect of the invention concerns the use of serum
(e.g., fetal bovine serum (FBS)), in SC cultures. It has already
been established that serum is a major source of undefined
differentiation factors and thus tends to promote ES cell
differentiation. Other problems are also associated with serum.
Lot-to-lot variation is often observed and some lots of serum have
been found to be toxic to cells [Robertson, E. J., ed.,
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,
IRL Press, Oxford, UK (1987)]. Moreover, serum may be contaminated
with infectious agents such as mycoplasma, bacteriophage, and
viruses. Finally, because serum is an undefined and variable
component of any medium, the use of serum prevents the true
definition and elucidation of the nutritional and hormonal
requirements of the cultured cells.
[0128] It has now been found that the use of two well known and
commercially available humanized serum replacement (SR)
substitutes, which have not been used hitherto in human embryonic
stem cell technology, i.e. TCH.TM. (Protide Pharmaceuticals, St.
Paul, Minn., or equivalent) and Nutridoma-CS (Roche, Germany, or
equivalent) used as serum replacement substitute, did not impair
the undifferentiated propagation of the stem cells in the culture
system.
[0129] Thus, according to a further aspect, the invention also
provide a humanized culture system for maintenance of stem cells
(SC) in an undifferentiated state, the humanized culture system
comprising animal free stem cell basic medium and a humanized serum
replacement substitute. This aspect of the invention is referred to
as the "humanized serum free culture system of the invention".
[0130] In accordance with one embodiment, the humanized culture
system comprises a serum free basic medium as known to those versed
in the art of stem cells (i.e. a medium which is free of animal
origin and is suitable for growth of stem cells), selected from
Cellgro Stem Cell Growth Medium, KO DMEM, Neurobasal.TM., or X-Vivo
10.
[0131] In accordance with another embodiment, the humanized culture
system comprises a serum replacement substituent selected from
TCH.TM., Nutridoma-CS or combination of same.
[0132] In accordance with yet another embodiment, when said serum
free basic medium is Neurobasal.TM., the culture system further
comprises N2 supplement [GIBCO.RTM. Cell Culture] or a modified N2
supplement, the modification rendering the medium supplement
suitable for use with stem cells. It is noted that the standard and
commercially available N2 supplement comprises insulin,
transferrin, progesterone, putrascine, selenite. The specific
composition of N2 supplement as published by StemCell Technologies
Inc (Product Information Sheet, revised on December 2002) includes
2.5 mg/mL rh insulin, 10 mg/mL human transferring (which may be
iron-poor or iron-saturated), 0.52 .mu.g/mL sodium selenite, 1.61
mg/mL putrascine, 0.63 .mu.g/mL progesterone, all in phosphate
buffered saline. Nonetheless, modifications of the standard N2
supplement for stem cells maintenance are readily envisaged by
those versed in the art. For example, medium for the propagation of
ESC-derived neural stem cells is supplemented with modified N2
[Conti, L, et al., Niche-independent symmetrical self-renewal of a
mammalian tissue stem cell. PLoS Biol. 9:e283 (2005)].
[0133] TCH.TM. is a completely biochemically defined serum
replacement developed primarily for human cells and production of
cell-secreted proteins. TCH.TM. may be purchased from Protide
Pharmaceuticals (MN, USA) as well as from BM Biomedicals (CA,
USA).
[0134] Nutridoma-CS is also a completely biochemically defined
serum free medium supplement composed of albumin, insulin,
transferrin, cytokines a cholesterol source and other defined
organic and inorganic compounds.
[0135] The use of humanized SR as suggested herein in combination
with serum free basic mediums thus enables the providence of an
animal free and humanized culture system for maintenance as well as
expansion of undifferentiated SC, preferably human SC, more
preferably, hESC. The culture system may comprise, in addition to
the humanized SR, human derived feeder cells such as that disclosed
above (as well as those known in the art),
[0136] The amount of the humanized SR may vary and will depend on
other elements forming part of the culture system. Those versed in
the art will know how to manipulate the concentrations of SR in the
culture system to facilitate maintenance of the stem cells cultured
therewith. According to one embodiment, 2% TCH may be used.
However, higher or lower concentrations of TCH may be used.
[0137] According to the humanized SR aspect of the invention there
is also provided the use of humanized serum replacement substitute
selected from TCH.TM., Nutridoma-CS or combination of same for the
preparation of a culture system for maintaining stem cells,
preferably human embryonic stem cells, in an undifferentiated
state. According to one embodiment, the cells are co-cultured with
feeder cells, preferably human derived feeder cells.
[0138] Finally, the invention provides a further culture system for
maintaining a stem cells, preferably human, more preferably, hESC,
in an undifferentiated state, the culture system comprising
Neurobasal.TM. medium. According to one embodiment, the
Neurobasal.TM. is supplemented with N2 supplement or a modification
of N2 supplement (as defined above) for a humanized flat culture
system of hESC on feeders (see for example FIG. 21) as well as for
maintenance of stem cells in suspensions (see for example FIG. 22).
Neurobasal.TM. is known in the art of cell cultures [Brewer G J.
Serum-free B27/Neurobasal medium supports differential growth of
neurons from the striatum, substantia nigra, septum, cerebral
cortex, J Neurosci Res. 42(5):674-83, (1995)] and is commercially
available [Gibco, Invitrogen cell culture, USA]. This aspect is
referred to herein as the "Neurobasal.TM. based culture aspect of
the invention".
[0139] As indicated above, and in accordance with one embodiment of
this aspect of the invention the culture system is supplemented
with N2 supplement, a chemically-defined additive for
Neurobasal.TM. Media.
[0140] Additional culture elements may be added, such as an extra
cellular matrix (ECM) which is a complex structural (network like)
entity surrounding and supporting cells, composed of different
combinations of the following three major classes of
bio-molecules:
[0141] Structural proteins: collagen and elastin.
[0142] Specialized proteins: e.g. fibrillin, fibronectin, and
laminin.
[0143] Proteoglycans: conjugates of a protein core and
glycosaminoglycans (GAGs).
[0144] Further additional elements may include growth factors, for
example, without being limited thereto, FGF (e.g. FGF2) as well as
others known in the art (see also FIGS. 23A and 23B).
[0145] Yet, additional elements which may be added include noggin
and activin A, as familiar to those versed in the art of stem
cells.
[0146] As appreciated by those versed in the art and also indicated
herein, there are some advantages in growing cells in suspensions.
For example, to exploit the potential of hESC for high throughput
screening, drug discovery, basic research, regenerative medicine,
and other potential applications, large numbers of cells are
required. The number of hESC that may be obtained with monolayer
cultures is limited. Culture of hESC in suspension rather than in a
monolayer is required to develop bulk cultures of hESC. Suspension
cultures of hESC may allow extensive expansion of the cells with a
bioreactor system. It may allow initiation of differentiation
processes in suspension of a large number of cells, and the
development of novel methodologies to direct differentiation of
hESC within suspension cultures.
[0147] In the context of this aspect of the invention there is also
provided a method for maintaining SC, preferably human, more
preferably, hESC, in an undifferentiated state, the method
comprising incubating the SC with a culture system comprising
Neurobasal.TM. in a growth environment, in which cells of interest
will proliferate in vitro, as detailed herein.
[0148] The invention will now be described by way of non-limiting
examples. It is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the examples. The invention is
capable of other embodiments or of being practiced or carried out
in other various ways. Also, it is to be understood that the
phraseology and terminology employed herein is for the purpose of
description and should not be regarded as limiting.
Some Exemplary Embodiments
Materials and Methods
[0149] Non-Clinical Grade Culture System of Human Feeders and
hESC
Human Feeders
[0150] Human foreskin or embryonic feeder cells were cultured in
DMEM (Gibco, Gaithersburg, Md.) supplemented with 10% Fetal Calf
Serum (Biological Industries, Beit Haemek, Israel). They were
passaged by Trypsin (Gibco, Gaithersburg, Md.) digestion and plated
on 0.1% Gelatin (Sigma, St. Louis, Mo.)-precoated tissue culture
dishes.
HES Cell Culture
[0151] Human ESC (HES1 and HES2 cell lines) were cultured on the
human feeder layers in KO medium (KOM) consisting of 85% KO-DMEM,
15% KO SR, 1 mM glutamine, 0.1 mM .beta.-mercaptoethanol, 1%
nonessential amino acids, 50 units/ml penicillin, 50 .mu.g/ml
streptomycin, (Gibco, Gaithersburg, Md.) and 4 ng/ml bFGF (R&D
Systems, Inc., Minneapolis, Minn.). hES cells were weekly passaged
by dissociation into nearly single cell suspension with
Ca/Mg.sup.++-free PBS supplemented with 0.05% EDTA (Biological
Industries, Beit Haemek, Israel) and plated onto fresh feeder
layer.
EBs Formation and Characterization
[0152] Human ES cells were removed from the feeders by treatment
with dispase (10 mg/ml; Gibco), and/or type IV collagenase (1
mg/ml; Gibco). The clusters of undifferentiated cells that were
obtained were further triturated into smaller clumps within PBS.
These clumps were cultured for various periods in suspension within
bacteriological dishes precoated with 0.1% low melting temperature
agarose in DMEM (Gibco), supplemented with 10-20% FCS (Biological
Industries, Beit Haemek), 1 mM L-glutamine, 0.1 mM
.beta.-mercaptoethanol, 1% non-essential amino acid stock, 50
units/ml penicillin, 50 .mu.g/ml streptomycin (all from Gibco
Invitrogen Corporation products, USA) in the presence or absence of
nicotinamide 10 mM (Sigma). In some experiments DMEM and FCS were
replaced by 86% KO-DMEM and 14% KOSR or by Neurobasal medium
supplemented by N2 (Gibco).
Immunohistological Studies of EBs
[0153] EBs that were developed 4 weeks in suspension culture in the
presence and absence of nicotinamide (NA), were fixed in 4%
paraformaldehyde at room temperature overnight. The EBs were then
concentrated at the bottom of a centrifuge tube and embedded in
agar. The agar blocks were dehydrated, and embedded in paraffin.
5-micron sections were immuno-stained with anti-human cytokeratin-8
and anti-.alpha.-fetoprotein (both at 1:50, monoclonal mouse IgG,
DAKO). Primary antibody localization was performed using
Picture.TM. plus kit (Zymed).
Indirect Immunofluorescent Staining of Differentiated Cells within
EBs or Neural Spheres
[0154] Differentiation into neural spheres was conducted according
to the published protocol (Itsykson, P., et al. Derivation of
neural precursors from human embryonic stem cells in the presence
of noggin. Mol Cell Neurpsci 30, 24-36 (2005). Spheres were
dissociated mechanically by trituration. Pigmented clusters of
cells within EBs, differentiating 8-10 weeks were mechanically
dissected by glass micropipettes or scalpel blades.
[0155] EBs were dissociated into smaller clusters mechanically
with/without the aid of trypsin (0.025%, 3 mM EDTA in PBS)
digestion. The small clusters of cells were plated on poly-D-lysine
(30-70 kDa, 10 .mu.g/ml; Sigma, St. Louis, Mo.) and laminin-coated
(4 .mu.g/ml; Sigma) glass coverslips and cultured for additional
3-5 weeks in the culture medium used for suspension culture of EBs
or neural spheres. Differentiated cells within the outgrowth were
fixed with 4% paraformaldehyde for 20 minutes at room temperature.
Cell membranes were permeabilized with 0.2% Triton X100 (Sigma) in
PBS for 5 minutes for immunostaining with ant-intracellular markers
antibodies. The cells were incubated with the following primary
antibodies: anti .beta.-tubulin III (mouse monoclonal IgG2b,
1:2000, Sigma), anti human alphafetoprotein (mouse monoclonal
IgG2a, 1:200, Sigma), anti human muscle actin (mouse monoclonal
IgG1, 1:10, Dako) and anti human desmin (mouse monoclonal
IgG1,.kappa., 1:10, Dako), anti-Pax6 (Developmental Studies
Hybridoma Bank; mouse monoclonal IgG.sub.1, 1:100), anti-MITF (Lab
Vision Corporation, Fremont, Calif.; mouse IgG.sub.1, 1:50),
anti-RPE65 (Novus Biologicals, Littleton, Colo.; mouse IgG.sub.1,
1:300), anti-Bestrophin (Novus Biologicals, Littleton, Colo.; mouse
IgG.sub.1, 1:150), anti-ZO-1 (Zymed Laboratories Inc., South San
Francisco, Calif.; rabbit polyclonal, 1:10) and anti-CRALBP (kindly
provided by John C. Saari, University of Washington, Seattle;
rabbit polyclonal, 1:100).
[0156] Primary antibody localization was performed by using
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse
immunoglobulins (Dako, 1:20-50), goat anti-mouse IgG conjugated to
Cy.sup.TH3 (1:500) and swine anti-rabbit Ig conjugated to
fluorescein isothiocyanate (FITC) (Dako, A/S Denmark; 1:50).
Analysis of EBs by RT PCR
[0157] For RT PCR analysis, total RNA was collected from EBs that
were developed 4 weeks in suspension culture in the presence and
absence of nicotinamide (NA). Total RNA was isolated using TRI
REAGENT (Sigma) and converted into complementary cDNA with M-MLV
reverse transcriptase (Promega). PCR was carried out using standard
protocols with Taq DNA Polymerase (Promega). Amplification
conditions were as follows: denaturation at 94.degree. C. for 60
seconds, annealing at 55-60.degree. C. for 60 seconds, and
extension at 72.degree. C. for 60 seconds. The number of cycles was
35 Primer sequences and lengths of amplified products were:
TABLE-US-00001 Chordine-like (NCBI Gene Bank: BC002909)
5'TGCAAGGTGTGTCCAGGTAA; (SEQ ID NO:1) 3'CCAGCTTGAAGTGAGGAAGC; (SEQ
ID NO:2)
[0158] The length of amplification product was 268 bp.
TABLE-US-00002 .alpha.-fetoprotein (NCBI Gene Bank NM001134)
5'CCATGTACATGAGCACTGTTG; (SEQ ID NO:3) 3'CTCCAATAACTCCTGGTATCC.
(SEQ ID NO:4)
[0159] The length of amplification product was 338 bp.
Intravitreal and Sub-Retinal Transplantation of hESC-Derived RPE
Cells
[0160] Clusters of pigmented cells were mechanically isolated by
scalpel blades from EBs that were differentiating in the presence
of NA for 6-8 weeks. The clumps were dissociated into smaller
clusters of cells by digestion with Papain (Papain Dissociation
System; Worthington Biochemical Corporation, Lakewood, N.J.) at
37.degree. C. for 30 minutes followed by trituration. Adult (body
weight 230-250 g) outbred RCS and albino rats were used for
intraocular transplantation. All animal experiments were conducted
according to the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research, and approved by the institutional
committee for animal research. The animals were anaesthetized with
Ketamine HCl (Ketalar, Parke Davis, UK, 100 mg/kg), injected
intra-peritoneally in combination with the relaxing agent Xylazine
(2.0 mg/kg). Local anaesthetic drops (Benoxinate HCl 0.4%, Fischer
Pharmaceuticals, Israel) were administered. The pupils were dilated
with Tropicamide 0.5% (Mydramide, Fisher Pharmaceuticals, Israel)
and Phenylephrine HCl 2.5% (Fisher Pharmaceuticals, Israel). Under
visualization of a dissecting microscope (Stemi SV 11, Zeiss,
Germany), about 100,000 cells in 4 .mu.L medium were injected into
the vitreous or to the subretinal space by a Hamilton syringe and a
33gauge needle via a transscleral, transchoroidal approach. Fellow,
non-injected eyes served as one type of control. As an additional
control, adult eyes were injected with saline (Sodium Chloride
Injection BP, 0.9%, B. Braun Melsungen AG, Melsungen, Germany).
During and after injection, no choroidal bleeding was observed.
Animals were kept warm throughout and after the procedure using a
heating lamp. Following transplantation, all animals received the
immunosuppressive agent cyclosporine A (Sandimmune, Novartis Pharma
AG, Basle, Switzerland, 50 mg/ml) in their drinking water at a
concentration of 210 mg/l. At 4 weeks post-injection, animals were
sacrificed and eyes enucleated for histological and
immunohistochemical examination. Following transcardial perfusion
with 4% paraformaldehyde in 0.1 M phosphate buffer, eyes were
embedded in paraffin and sectioned at 4 .mu.m serial sections. Each
fifth slide was stained with hematoxylin and eosin for
histomorphologic evaluation. For indirect immunofluorescent
studies, specimens were de-parafinized in xylene and dehydrated in
graded alcohols, rinsed with phosphate-buffered saline (PBS, pH
7.4), and incubated with 10 mM citrate buffer (pH 6.0) at
110.degree. C. for 4 minutes. After washing with PBS, specimens
were blocked for 1 hour at room temperature with PBS solution
containing 1% bovine serum albumin, 0.1% triton-.times.100, and 3%
normal goat serum. Subsequently, sections were incubated for 24
hours at 4.degree. C. in a humidified chamber with anti-green
fluorescent protein (anti-GFP; Santa Cruz Biotechnology; rabbit
polyclonal, 1:100). After washing in PBS, specimens were incubated
for 1 hour at room temperature with Cy.TM.2-conjugated goat
anti-rabbit IgG (1:200). Nuclei were counterstained with 4,
6-diamidino-2-phenylindole (DAPI)-containing mounting medium
(Vector Laboratories, Inc., Burlingame, Calif.). To determine the
specificity of the antigen-antibody reaction, corresponding
negative controls with an irrelevant isotype-matched antibody were
performed. A Zeiss Axiovert 200 microscope equipped with Sensi Cam
12 Bit imaging (Zeiss, Kelheim, Germany) was used for fluorescent
and light microscopy imaging.
Clinical Grade Feeder and hESC Culture System
Development of Human Feeders
[0161] Human feeders were obtained via Informed Consent from
aborted fetuses (10-12 weeks of gestation), umbilical cords, and
newborn foreskin.
[0162] The legs and hands of aborted human fetuses were washed
twice and transferred to a drop of 20011 of medium comprised of HyQ
DMEM (Hyclone, Utah) containing 10% human serum (Cambrex,
Maryland,) and were minced with two scalpel blades into small
pieces of tissue. The minced embryonic tissues were incubated 10
minutes in 10 ml TrypLE Select (Gibco, Gaithersburg, Md.) solution
at 37.degree. C. with intermittent shaking. The supernatant was
then collected and inactivated by diluting the solution with 35 ml
of the medium for fibroblast culture (feeders medium) that was
comprised of HyQ DME (Hyclone, Utah) supplemented with 10%-20%
human serum (Cambrex, Maryland), and 2 mM glutamine (Hyclone,
Utah). This procedure was performed twice. The cells were spun down
and 4-5.times.10.sup.6 cells were plated in T-25 tissue culture
dishes for further propagation in feeders medium as above. When
confluent, they were passaged using TrypLE Select solution (Gibco,
Gaithersburg, Md.).
[0163] Term umbilical cord tissue was minced as above, and the
small pieces of tissue were plated in the fibroblast culture
medium, as described above. To promote the adherence of the tissue
pieces to the culture dish, flasks were incubated upright
overnight. Cells emanating from the tissue pieces were propagated
as above.
[0164] Foreskin tissue was obtained from 7 day to 6 months
circumscribed newborns and babies. Circumcision was performed in
the operating room, and the medium described above was used for the
development and culture of the foreskin feeders. The circular
foreskin was cut and spread on the tissue culture dish. The
epidermis was scraped with scalpel blade followed by washing with
the culture medium. This was repeated 5 times and small foreskin
tissue pieces were plated in the culture medium within a flask. The
flask was incubated upright to enhance the adherence of tissue
pieces to the dish and promote outgrowth of fibroblasts.
[0165] All fibroblasts were cryopreserved in human serum (Cambrex,
Maryland, Maryland) supplemented with 10% Cryosure-DMSO
(Wak-Chemie, Germany). Slow-cooling and rapid-thawing standard
methods were used.
[0166] For indirect immunofluorescent staining the fibroblasts were
plated and cultured on cover slides. They were fixed with 4%
paraformaldehyde for 20 minutes at room temperature, and then
permeabilized with 0.2% Triton X100 (Sigma) in PBS for 5 minutes.
The cells were incubated with anti human vimentin (mouse monoclonal
IgG2a, .kappa. Dako, 1:10). Primary antibody localization was
performed by using fluorescein isothiocyanate (FITC)-conjugated
goat anti-mouse immunoglobulins (Dako, 1:50).
[0167] FACS analysis of marker expression by the feeders was
performed by using a FACS Calibur system (Becton-Dickinson, San
Jose, Calif.). Propidium Iodide was added (final concentration of 4
.mu.g/ml) for better gating of viable cells. The feeders were
disaggregated using TrypLE Select solution (Gibco, Gaithersburg,
Md.). The cells were, then washed with FACS media consisting of PBS
supplemented with 1% BSA and 0.05% sodium azide. The single cell
suspension was stained with
[0168] Anti-human fibroblasts antibodies (mouse monoclonal IgG2a,
Acris, 1:100) and Anti human CD44 FITC-conjugated antibody (mouse
monoclonal IgG2b, IQ Products, The Netherlands, 1:10). Control
feeders were stained with an isotype control antibody. Primary
anti-human fibroblasts antibodies were detected with a FITC-labeled
goat anti-mouse Ig (1:100, Dako).
Clinical Grade hESC Culture System
[0169] Human embryonic fibroblasts derived from umbilical cord as
described herein or foreskin fibroblasts were used as feeders. The
feeders were plated in tissue culture dishes precoated with 1
.mu.g/cm.sup.2 human Fibronectin (BD Biosciences, Bedford, Mass.)
or 100 .mu.g/ml recombinant Gelatin (FibroGen, SF). Mitotic
inactivation was carried out by incubating the feeders 2.5 hours
with Mitomycin-C (Kyowa, Tokyo). KO DMEM (Gibco), X-Vivo 10
(Biowhittaker, Maryland), or Cellgro Stem Cell Growth Medium
(CellGenix Freiburg, Germany) were used as the basic media. They
were supplemented with TCH (1-2%; Protide Pharmaceuticals, St.
Paul, Minn.) or Nutridoma-CS (2%; Roche, Germany) as serum
substitutes. The media were supplemented with 2 mM glutamine
(Hyclone, Utah) and non-essential amino acids (NEAA, 1%; Hyclone
Utah). hESC were split weekly with Ca/Mg.sup.++-free PBS
supplemented with 0.05% EDTA.
[0170] For cryo-preservation, hESC were disaggregated with
Ca/Mg.sup.++-free PBS supplemented with 0.05% EDTA, spun down, and
re-suspended in Cellgro medium, supplemented with 2% TCH, and 10%
Cryosure-DMSO (Wak-Chemie, Germany). Conventional slow-rate cooling
and rapid-thawing methods were used.
[0171] FACS analysis of marker expression was performed on hESC
after disaggregation using Ca/Mg.sup.++-free PBS supplemented with
0.05% EDTA. The cells were then washed with FACS media consisting
of PBS supplemented with 1% BSA and 0.05% sodium azide. The single
cell suspension was stained with anti-SSEA4 (1:100, mouse
monoclonal IgG3, Developmental Studies Hybridoma Bank (DHSB), Iowa
City, Iowa), and anti-Tra-1-60 (1:20, monoclonal mouse IgM, gift
from Prof. P. Andrews), anti-Tra-1-81 (1:100, monoclonal mouse IgM,
Chemicon International), anti-human Oct 4 (mouse monoclonal IgG2b,
1:50, Santa Cruz), and anti-SSEA1 (1:100, Chemicon). Control hESC
were stained with the respective isotype control antibodies.
Primary antibodies were detected with a FITC-labeled goat
anti-mouse Ig (1:100, Dako).
Suspension Culture of hESC
[0172] In the course of the routine passaging procedure, hESC
colonies were dissociated with 0.05% of EDTA for 7-10 min at
37.degree., or mechanically with the aid of collagenase IV (1
mg/ml, 120 min at 37.degree.). Cell dissociation was promoted by
gentle blowing of the medium with a 1 ml pipette tip towards the
hESC colonies. The cells/cell-clusters were re-suspended in NBN2
medium (Neurobasal, N2 supplement 1:100, glutamine 2 mM, 50
units/ml penicillin, 50 .mu.g/ml streptomycin) supplemented with
bFGF 20 ng/ml, noggin 250 ng/ml, activin 25-50 ng/ml, fibronectin
and laminin 5 ng/ml each, gelatin 0.001%. The suspension may be
strained though 30-50 micron mesh to remove big clumps and
transferred into tissue culture dishes (Costar.RTM., Corning Inc.,
Corning, N.Y., USA) at a density of .about.0.7-1.2.times.10.sup.6
cells/ml. Dead cells and their fragments were gradually removed
during media refreshment and small transparent cell aggregates
emerged from the 3.sup.rd-5.sup.th days of suspension culture. The
cells proliferated as free-floating tiny clusters of 20-50 cells.
Aggregation and overgrowth of clusters was prevented by their daily
trituration with a 1000 .mu.l pipettor tip. Alternatively, every 7
days clusters were dissociated with Ca/Mg-free PBS supplemented
with 3 mM EDTA and 0.06%_trypsin and the single cells were
resuspended in fresh medium.
Characterization of hESC Grown in Suspension
[0173] For characterization of the cells within the small floating
aggregates, they were dissociated with EDTA solution (as above),
followed by gentle trituration, and were plated in NBN2 on glass
coverslips, pretreated with poly-D-lysine (30-70 kDa, 10 .mu.g/ml;
Sigma, St. Louis, Mo.) and laminin (4 .mu.g/ml; Sigma). After one
hour's incubation at 37.degree., the cells were incubated 1 min in
propidium iodide solution (PI, 1 g/ml in PBS, Sigma) in order to
distinguish dead cells from vital ones. The cells were then washed
with PBS and fixed with 4% PFA for 20 min. For immunostaining with
antibodies against surface markers, the fixed cells were incubated
for 30 min at room temperature (RT) with the following primary
antibodies: anti-SSEA4 (1:200 of the concentrated monoclonal mouse
IgM, Developmental Studies Hybridoma Bank (DHSB), Iowa City, Iowa),
anti-Tra-1-60 (1:20), anti-Tra-1-81 (1:10) (monoclonal mouse IgM,
gift from Prof. P. Andrews), anti-GCTM2 (supernatant, monoclonal
mouse IgM, gift from Prof. M. Pera). For nuclear marker
characterization, cells were pre-treated with 0.2% Triton X-100
(Sigma) and then stained with anti-Oct-4 (1:100, mouse monoclonal
IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). Primary
antibody localization was performed using fluorescein
isothiocyanate (FITC)-conjugated with either goat anti-mouse
immunoglobulins (Dako, 1:20) or goat anti-mouse IgM (1:200, Jackson
Immunoresearch Laboratories, Inc., West Grove, Pa.). For the
characterization of the effects of various growth factors and ECM
components on the suspension culture system, hESC with an extra
short arm (p) of chromosome 1 were used. FACS analysis was
performed as above.
Results
[0174] Derivation and Propagation of hESC in Animal-Free Clinical
Grade Culture Systems.
[0175] The development of animal-free culture systems for the
propagation of undifferentiated hESC is an essential step towards
the exploitation of hESC in transplantation therapy. To this end,
culture systems for the derivation of human feeder cell and hESC
lines from which therapeutic products can be derived have now been
developed. These systems are compliant with both FDA and European
regulations for Biological Products, using good manufacturing
practices (GMPs) in their manufacture, good tissue practices (GTPs)
in their production, and good laboratory practices (GLPs) in their
testing. In these culture systems, animal-derived reagents are not
used, and all reagents are purchased from sources that are
GMP-compliant.
Human Feeder Cells (Feeders)
[0176] Various human feeders may be utilized, in these systems, for
the derivation and propagation of undifferentiated hESC, such as
human embryonic fibroblast (HEF) feeders, umbilical cord derived
fibroblast feeders and foreskin-derived fibroblasts.
[0177] In the examples provided herein the feeder cells were
developed and cultured using a clinical grade animal free culture
system comprising the following reagents: [0178] DMEM (HyQ DME
(Hyclone, Utah or equivalent)); [0179] Human serum (Cambrex,
Maryland, or equivalent), replacing fetal calf serum (FCS), which
is most commonly used; [0180] Human fibronectin or recombinant
gelatin (FibroGen, SF, or equivalent), replacing the commonly used
porcine gelatin for tissue culture dish coating; [0181]
TrypleSelect (Gibco, Gaithersburg, Md., or equivalent), a
recombinant enzyme, replacing animal-derived trypsin for splitting
of fibroblasts; [0182] Cryosure-DMSO (Wak-Chemie, Germany), or
equivalent, a GMP-qualified product for fibroblast
cryopreservation; [0183] Mitomycin-C (USP or equivalent) from a
GMP-qualified source for fibroblasts mitotic inactivation.
[0184] By using the above reagents, new human fibroblast cell lines
were derived from foreskin, aborted fetuses and umbilical cords
(FIG. 1A-1D).
[0185] Overall, 8 new foreskin-derived, 10 umbilical cord-derived
and 5 aborted fetuses-derived fibroblast cell lines were developed,
of which 6, 8, and 3 were developed within a GMP facility,
respectively.
[0186] Two fibroblasts cell lines derived from each of the three
groups (overall 6 cell lines), were characterized between passages
5-8. The cells had a typical fibroblast morphology (FIGS. 1A and
1C). Immunostaining demonstrated that over 70% of the cells in each
of the cell lines were immunoreactive with markers of fibroblasts
including but not limited to anti-vimentin (FIGS. 2A-2F), anti-CD
44 and anti-human fibroblasts antibody (FIG. 3). The karyotype of
the feeders was normal (FIG. 4A). Fibroblasts doubling rate was
between 20-50 hours (Table 1). The DNA had a definitive human STR
profile (not shown).
TABLE-US-00003 TABLE 1 Number of umbilical cord derived fibroblast
cells Time = Time = Time = Time = Doubling Feeder Line 0 24 hr. 48
hr. 75 hr. Time Cord 1 P.sub.6* 118148 252777 330833 400000 42.3
hrs Cord 2 P.sub.6 35185 52962 83555 91851 49.9 hrs Foreskin 2
P.sub.5 31705 130000 260000 273000 23.2 hrs Foreskin 4 P.sub.5
36666 115000 232592 396111 21.2 hrs HEF1 P.sub.6 54259 114259
176876 357916 27.3 hrs HEF2 P.sub.6 61093 115370 199092 309166 31.8
hrs *P.sub.i denotes the number of passages
[0187] In order to evaluate the potential of these fibroblast cell
lines to support the undifferentiated propagation of pluripotent
hESCs, hESCs were cultured on the feeder layers for a period of ten
weeks and their phenotype as well as developmental potential were
characterized. The phenotype of the hESCs was characterized at two
time periods. The first time point, between passages 1-5 and the
second time point, between passages 6-10.
[0188] Human embryonic stem cells that were propagated on the three
types of feeders (after freezing and thawing of the feeders)
maintained their typical morphology (FIGS. 5A-5C) and expressed the
following cell surface markers of undifferentiated hESCs: SSEA-4,
TRA-1-60, TRA-1-81 (FIG. 6A-6L) alkaline phosphatase (AP; FIGS.
7A-7C), and Oct-4 (FIG. 8A-8F). Above 70% of the hESCs expressed
markers of pluripotent cells when analyzed at the early and late
passage periods (FIGS. 9-11, hESCs on cord, foreskin and HEF
fibroblasts respectively; FIGS. 12-14 for Oct4, respectively). The
percentage of hESC expressing SSEA1, a marker of differentiated
cells, was less than 15% (FIGS. 9-11). These data indicated that
all types of fibroblasts could support undifferentiated
proliferation of the hESCs.
[0189] Doubling time of hES cultured on the feeders was 21-30
hours, with one exception of 36 hours (Table 2).
TABLE-US-00004 TABLE 2 No. of hESC cultured on three different
types of feeders. Time = Time = Time = Time = Doubling Feeder Line
48 hr 120 hr 144 hr 168 hr Time hESC on 26666 85185 172222 271851
35.8 hrs Cord 1 hESC on 28148 15666 397778 703166 20.3 hrs Cord 2
hESC on 25555 192251 275925 52000 28 hrs Foreskin 2 hESC on 26667
58518 493611 790000 19.4 hrs Foreskin 4 hESC on 72129 448055
1147778 -- 25.4 hrs HEF1 hESC on 84445 593612 1098889 -- 27 hrs
HEF2
[0190] In these experiments, the hESC were cultured in KO DMEM
supplemented with 20% KO SR.
[0191] In vitro differentiation of hESC cultured on the various
feeder layers was induced by the derivation of EBs and
neurospheres. The EBs and neurospheres were dissociated, plated,
and stained for markers of the three germ layers (Alfafetoprotein
for endoderm, .beta. Tubulin III for ectoderm, and muscle-desmin or
muscle actin for mesoderm). Human embryonic stem cells that were
propagated for 10 passages on each of the three types of feeders
could differentiate in vitro into progeny representing the three
germ layers (FIG. 15A-15I). These data show that the developmental
potential of the hESC, when propagated on the three types of
feeders, could be maintained for extended periods of time in
culture.
[0192] The cord-derived feeders could be maintained for prolonged
periods (17 passages). It has also now been found that
proliferation of the fibroblasts could be augmented by
supplementation of the medium with FGF2 (FIG. 16). The fibroblasts
maintained their typical morphology and marker expression following
expansion with or without FGF2 supplementation (FIG. 17A-17D).
Cord-derived fibroblasts that were propagated in culture for
extended periods of time with or without the addition of FGF2 could
support undifferented proliferation of hESC (FIG. 18A-18G).
[0193] The feeder cells from the three types of primary tissues
could be successfully cryopreserved and thawed. Above 55% of the
cells survived thawing. The morphology and marker expression and
growth rate were not altered by cryopreservation. Cryopreserved
feeders from the three sources could support undifferentiated
proliferation of pluripotent hESC, as detailed above. When the
feeder cells were cryopreserved with our usual cryopreservation
solution (knockout (KO) DMEM, 10% DMSO, 10% KO serum replacement
(SR)), or in solutions that do not include animal components (90%
Human Serum and 10% DMSO), cell viability and growth rate
post-thawing were similar with both cryopreservation solutions.
Human Embryonic Stem Cells
[0194] Animal-free culture system for hESC employed in the
exemplary embodiments presented here, included the following
components: [0195] KO DMEM (Gibco, or equivalent), X-Vivo 10
(Biowhittaker, Maryland, or equivalent) or Cellgro Stem Cell Growth
Medium (CellGenix, Freiburg, Germany, or equivalent) used as the
basic media. [0196] Humanized serum substitutes TCH (Protide
Pharmaceuticals, St. Paul, Minn., or equivalent) or Nutridoma-CS
(Roche, Germany, or equivalent) used as serum replacement
substitute. [0197] Human fibronectin (BD Biosciences, Badford,
Mass., or equivalent) or recombinant gelatin (FibroGen, SF, or
equivalent), used for tissue culture dish coating. [0198] PBS
without calcium and magnesium, supplemented with 0.05% EDTA used
for splitting. [0199] Cryosure-DMSO (Wak-Chemie, Germany, or
equivalent) used for cryopreservation.
[0200] KO DMEM, X-Vivo 10, or Cellgro Stem Cell Growth Medium were
used as the basic media for the propagation of undifferentiated
hESC colonies on Human Embryonic Fibroblast (HEF) or foreskin
feeders. These basic media were supplemented with TCH, which is an
animal-free, defined serum replacement (SR) substitute. TCH
replaced KO SR, which contains animal products and which is most
commonly used as a supplement to KO medium.
[0201] Undifferentiated proliferation of hESC was obtained with TCH
supplementing all three basic media. With 2% TCH in KO DMEM, 88% of
the hESC that were cultured on foreskin feeders expressed SSEA-4.
With 1% TCH supplementing Cellgro medium, 98% of the cells were
SSEA-4+, and with 2% TCH in X-Vivo 10 medium, 99%, respectively.
High percentages (86%) of the hESC also expressed the marker
TRA-1-60, when propagated on HEF in Cellgro with the addition of 1%
TCH. The hESC retained the morphology of undifferentiated
pluripotent stem cells, when cultured in media supplemented with
TCH (FIG. 19).
[0202] In addition to TCH, it has now been found that Nutridoma-CS,
which is animal free, can also serve as a SR substitute for the
support of undifferentiated propagation of hESC. Both TCH and
Nutridoma-CS were found to be as effective as KO SR in supporting
undifferentiated propagation of hESC. When hESC were cultured on
human feeders (embryonic fibroblasts or foreskin) in KO DMEM
supplemented with KO SR, 2% TCH, or 2% Nutridoma-CS, the percentage
of cells expressing SSEA-4 was comparable in the three systems. The
percentages were 93% and 79% with KO+ SR on Foreskin and HEF,
respectively; 97% and 72% with KO+ TCH, and 93% and 86% with KO+
Nutridoma-CS (FIG. 20).
[0203] In the herein disclosed animal-free system, human
fibronectin or recombinant gelatin replaced the animal gelatin that
is most commonly used as a matrix to pre-coat the tissue culture
dishes. With regard to recombinant gelatin, when hESC were cultured
on a foreskin feeder layer in KO DMEM+KO SR on culture plates
precoated with gelatin or recombinant gelatin, the percentage of
SSEA-4+ hESC was comparable (91% and 94%, respectively). With
regard to human recombinant fibronectin, when hESC were cultured in
Cellgro medium supplemented with 2% TCH on a matrix of gelatin or
human fibronectin, the percentage of SSEA-4+ cells was 97% and 96%,
respectively, and the percentage of Tra-1-60+ cells was 92% on both
matrices.
[0204] In herein disclosed humanized system,
Penicillin/Streptomycin and .beta.-Mercaptoethanol were omitted
from the hESC culture media without affecting the growth rate or
the level of background differentiation.
[0205] For cryopreservation of USC, Cryosure-DMSO from Wak-Chemie,
Germany, a GMP-compliant company, was successfully used. When hESC
were cryopreserved in Cellgro medium supplemented with 2% TCH, and
10% Cryosure-DMSO, 2 weeks after thawing and culturing on foreskin
fibroblast feeders, the percentage of hESC expressing SSEA-4 and
TRA-1-60 was 98% and 95%, respectively. These results were
comparable to those obtained with our usual freezing solution
comprised of 90% FCS and 10% DMSO(SSEA-4 98% and TRA-1-60 96%).
[0206] In an alternative system, Neurobasal.TM. (NB) medium, which
was initially developed for the maintenance of neural cells in
ambient atmosphere, and is animal-reagent free, is used as the
basic medium of the culture system. When supplemented with N2
supplement (which includes insulin, transferrin, progesterone,
putrascine, selenite, NBN2) it allows undifferentiated
proliferation and propagation of hESC as flat colonies on the
feeders. The hESC are weekly sub-cultured following mechanical or
enzymatic (dispase, type IV collagenase, or trypsin) disaggregation
or treatment with Ca/Mg.sup.++-free PBS supplemented with EDTA, or
the combination of the above. The hESC retained the morphology of
undifferentiated cells (FIG. 21), and expression of alkaline
phosphatase (AP). The medium may be supplemented with FGF2.
Suspension Culture System for the Propagation of hESC in Bulk
[0207] To exploit the potential of hESC for high throughput
screening, drug discovery, basic research, regenerative medicine,
and other potential applications, large numbers of cells are
required. The number of hESC that may be obtained with monolayer
cultures is limited. Culture of hESC in suspension rather than in a
monolayer is required to develop bulk cultures of hESC. Suspension
cultures of hESC may allow extensive expansion of the cells with a
bioreactor system. It may allow initiation of differentiation
processes in suspension of a large number of cells, and the
development of novel methodologies to direct differentiation of
hESC within suspension cultures.
[0208] Propagation of hESC in suspension is achievable using
Neurobasal.TM. medium as the basic medium of the culture system.
The Neurobasal.TM. medium may be supplemented with N2 (NBN2). It
may be further supplemented with soluble factors including but not
limited to FGFs, TGF.beta. superfamily factors, BMPs antagonists as
well extracellular matrix (ECM) components including but not
limited to fibronectin, laminin, gelatin to promote proliferation
and survival of the cells and to prevent their differentiation.
[0209] To develop suspension cultures, hESC colonies that are
cultivated on human feeders in the KO culture system are
dissociated with the aid of type IV collagenase or
Ca/Mg.sup.++-free PBS supplemented with 0.05% EDTA. The cells/cell
clusters that are obtained are re-suspended within fresh NBN2
medium, supplemented with FGFs (bFGF 20 ng/ml).
[0210] Further supplementation of the medium with the following
components increases the survival/proliferation and prevent
differentiation of the cells: [0211] TGF.beta. superfamily factors
(activin 25-50 ng/ml) [0212] BMPs antagonists (noggin 250 ng/ml)
[0213] ECM components (laminin 5 ng/ml, fibronectin 5 ng/ml,
gelatin 0.001%)
[0214] The cells are transferred to suspension at a density of
.about.0.7-1.2.times.10.sup.6 cells/ml. Dead/fragmented cells are
gradually removed during medium refreshment. After 3-5 days of
suspension culture, small transparent cell-aggregates develop.
These transparent cells proliferate as free-floating tiny clusters
of 20-50 cells without any morphological signs of differentiation
(FIG. 22A). Aggregation and overgrowth of the transparent clusters
may be prevented by daily trituration through a 1000 .mu.l pipette
tip or by the use of bioreactor. The cells express SSEA4 and OCT4,
(FIGS. 22B and 22D respectively). When re-plated on human feeders,
after 6 weeks of suspension culture, they give rise to colonies
with the morphology of undifferentiated hESCs.
[0215] It was further found and disclosed herein that when the
hESCs are cultivated 3 weeks in suspension under these culture
conditions in medium supplemented with all the above mentioned
components, .gtoreq.85% of the cells express SSEA4. Thus, these
culture conditions can support undifferentiated cultivation of hESC
in suspension.
[0216] Analysis of the effect of each of the components, in the
presence of FGF2, showed that ECM components promoted
survival/proliferation of hESCs (FIG. 23B Lam=laminin,
Fn=fibronectin, Gel=gelatin), activin A prevented differentiation
(FIG. 23A), and noggin could promote survival/proliferation (FIG.
23B).
[0217] Nicotinamide may be added to the medium to prevent the
differentiation of the cells towards extraembryonic lineages, to
promote their survival and to maintain them undifferentiated.
Nicotinamide (NA) for the Maintenance of Undifferentiated hES
Cells, Prevention of Extraembryonic Differentiation and for the
Induction of Somatic Differentiation.
[0218] The novel effect of NA on hESCs' differentiation was most
profoundly demonstrated during spontaneous differentiation of hESCs
within embryoid bodies (EBs). When hESCs differentiate within
free-floating clusters, in serum-supplemented medium, with time
they develop into cystic EBs which include cystic structures and
areas of more densely packed cells (FIGS. 24A-24B). These cystic
structures are attributed in mouse EBs to extraembryonic endodermal
differentiation [Coucouvanis, E. & Martin, G. R. Signals for
death and survival: a two-step mechanism for cavitation in the
vertebrate embryo. Cell 83, 279-287 (1995); Doetschman, T. C., et
al. The in vitro development of blastocyst-derived embryonic stem
cell lines: formation of visceral yolk sac, blood islands and
myocardium. J Embryol Exp Morphol 87, 27-45 (1985)]. However, in
the presence of NA, preferably at a concentration of 10 mM, cystic
structures were not formed during differentiation within EBs (FIGS.
24C-24D). Moreover, the differentiated cells acquired the
morphology of neural precursor cells.
[0219] The effect of NA on differentiation within EBs was not
dependent on the presence of serum and was observed also when EBs
differentiated in a chemically-defined medium (NBN2; FIGS. 25A-25H,
i.e. upper two panels).
[0220] In addition to its effect on differentiation, NA also had an
effect on the size of the EBs. In serum-free medium supplemented
with NA, the size of the Ebs after two weeks of differentiation was
significantly higher (1.7 times; maximal diameter of EBs) in
comparison to the size of EBs that were cultured under the same
conditions in the absence of NA (FIGS. 25E-25F, i.e. left pictures
of upper two panels). The increased size of the EBs in the presence
of NA reflected an increase in the number of cells within the EBs.
When EBs were generated from a comparable number of
undifferentiated cells, after 2 and 4 weeks, there were 5.4 and 33
times more cells respectively, in cultures that were treated with
NA as compared to controls. Thus NA can be used to increase the
number of differentiated cells that are obtained from hESC.
[0221] Furthermore NA treatment allowed the prolonged culture of
EBs for 12 weeks (FIG. 28). Prolonged culture of EBs is important
for the completion of complex differentiation processes that
require long time periods, and may promote the maturation of
differentiated cells.
[0222] Retinoic acid is known to induce extraembryonic
differentiation of human pluripotent stem cells (Roach, S., Schmid,
W., Pera, MF. Hepatocytic transcription factor expression in human
embryonal carcinoma and yolk sac carcinoma cell lines: expression
of HNF-3 alpha in models of early endodermal cell differentiation.
Eep Cell Res 215, 189-98 (1994). When hESC-derived EBs were
cultured in the presence of RA there was extensive cystic
formation. NA could block the effect of RA and when EBs were
cultured in the presence of RA and NA the development of cystic
structures was infrequent (FIGS. 251-25P, i.e. lower two
panels).
[0223] In addition, the number of differentiated cells that were
obtained in the presence of RA was low. This effect of RA was also
blocked when the medium was supplemented with NA (FIGS. 25I-25P,
lower two panels).
[0224] Immunocytochemical and RT-PCR studies showed that the
expression of the endodermal (extraembryonic and definitive) marker
.alpha.-fetoprotein, which is expressed by cells within EBs that
are cultured in the presence of serum, was not detected within the
EBs following differentiation in the presence of NA (FIGS. 26 and
27A, 27C). Similarly, Cytokeratin-8, which is a nonspecific marker
of a wide range of epithelial tissues, including extraembryonic
tissues [Pera, M. F. et al. Regulation of human embryonic stem cell
differentiation by BMP-2 and its antagonist noggin. J Cell Sci 117,
1269-1280 (2004); Kemler, R., et al. Reactivity of monoclonal
antibodies against intermediate filament proteins during embryonic
development. J Embryol Exp Morphol 64, 45-60 (1981)], and which is
widely expressed within EBs, was not demonstrated within EBs that
were developed in the presence of NA (FIGS. 27B and 27D).
[0225] A high percentage of the cells within EBs that
differentiated in the presence of NA (for 6 weeks) expressed neural
precursor markers in comparison to control EBs that were developed
in the absence of NA (PSA-NCAM 85.5% vs. 19.9% respectively, FACS
analysis). In addition, indirect immunofluorescence studies showed
that in the presence of NA, 58.3% of the cells expressed nestin,
and 67.8% expressed .beta.-tubulin III.
[0226] Within the neural lineage, NA treatment was found to promote
differentiation towards RPE cells. RPE cells, which lie immediately
underneath the photoreceptors and form part of the retina-blood
barrier towards the choroid, play a crucial role in supporting and
maintaining the photoreceptors. Their tasks include active
transport of nutrients from the choroidal vessels to the
photoreceptors, processing of vitamin A, and uptake and recycling
of outer segments which are continuously shed by the
photoreceptors. While primary degeneration of the photoreceptors is
the cause of progressive visual loss in some types of Retinitis
Pigmentosa, in others the initial injury is in RPE cells, and as a
consequence, the photoreceptors are damaged as well and retinal
degeneration ensues. Even more importantly, in Age-Related Macular
Degeneration (AMD), which is the most common cause of blindness
among the elderly aging population, failure of the RPE is the main
cause of disease [Smith W, Assink J, Klein R et al. Risk factors
for age-related macular degeneration: Pooled findings from three
continents. Ophthalmology 2001; 108:697-704.
[0227] Thus, derivation of this cell type from hESC may be of great
benefit. They may be used as an in vitro model for the development
of new drugs to promote their survival and function. hESC-derived
RPE cells may serve for high throughput screening for compounds
that are toxic to RPE cells. They may be used to uncover
mechanisms, new genes, soluble or membrane-bound factors that are
important for the development, differentiation, maintenance,
survival and function of photoreceptor cells. These cells may serve
as an unlimited source of RPE cells for transplantation and
replenishment of malfunctioning or degenerated RPE cells in retinal
degenerations. Genetically modified RPE cells may serve as a vector
to carry and express genes in the retina after transplantation.
[0228] While pigmented cells were rarely observed within
hESC-derived EBs 1 that were differentiating in KO medium, clusters
of pigmented cells were abundant within EBs differentiating in the
presence of NA. The percentage of EBs that included clusters of
pigmented cells gradually increased along differentiation in the
presence of NA (34%, 59% and 70% at 5, 6 and 12 weeks of
differentiation respectively; FIGS. 28A-28D. After 6 weeks of
differentiation, 9% of the cells within the EBs were pigmented. The
potential of NA to induce differentiation towards RPE cells was
also demonstrated at the RNA level by Real Time PCR. The expression
of the RPE markers RPE65 and MITF A was significantly increased in
EBs that differentiated in the presence of NA compared to control
EBs (FIG. 29).
[0229] When the EBs were partially dissociated and plated, colonies
that were comprised of a monolayer of the pigmented cells were
formed between other types of differentiated cells (FIGS. 30A-30H).
Indirect immunofluorescent staining showed that the pigmented cells
expressed multiple markers of RPE cells including PAX6, MITF, ZO-1,
CRALBP, Bestrophin and RPE65 thus confirming the retinal identity
of the pigmented cells.
[0230] It was possible to enrich the cultures for the RPE cells by
isolating the pigmented cells from other cells within the EBs by
mechanical dissection. Other methods such as genetic selection,
immunopanning, FACS and others may be used to purify the cultures
for RPE cells.
[0231] Enriched populations of hESC-derived RPE cells were
transplanted into the vitreous and into the subretinal space of RCS
and albino rats respectively. Immuno-histological analysis of the
eyes, at 4 weeks after transplantation, demonstrated surviving
hESC-derived RPE cells (FIG. 32A-32B). Transplanted RPE cells
migrated from subretinal grafts and integrated within the RPE layer
of host albino rats (FIG. 32C).
[0232] A differential comparison of the gene expression profile of
EBs after 4 weeks of suspension culture in the presence and absence
of NA was performed by using affymetrix gene arrays. The expression
level of 1072 genes was up- or down-regulated by at least two-fold;
442 genes were up-regulated, and 630 genes were down-regulated. The
analysis confirmed that NA suppressed the expression of
.alpha.-fetoprotein and cytokeratin-8. In addition, the expression
of other cytokeratins was also suppressed, including cytokeratins
7, 18, 19, 23.
[0233] Interestingly, the expression of chordin-like-1 gene was
up-regulated. This finding was further confirmed by RT PCR (FIG.
31). Chordin-like is composed of three cysteine-rich (CRs) domains.
Chordin-like binds BMP-4, BMP-5, BMP-6 and TGF-.beta.1&2
[Nakayama, N. et al. A novel chordin-like protein inhibitor for
bone morphogenetic proteins expressed preferentially in mesenchymal
cell lineages. Dev Biol 232, 372-387 (2001)]. In the early mouse
embryo, chordin expression is restricted to the node and the
notochord at a time in which chordin-like is expressed in the
neural plate [Garcia Abreu, J., et al. Chordin-like CR domains and
the regulation of evolutionarily conserved extracellular signaling
systems. Gene 287, 39-47 (2002)]. NA treatment induced the
expression of genes that are involved in active Wnt signaling
pathway including Wnt4, Wnt 2B, Frizzled (FZD) 1, FZD2, FZD3,
FZD10.
[0234] In line with the morphological and immunostaining findings
that showed the potential of NA to promote neural differentiation,
the expression of ZIC1, a zic family member 1, that is expressed
during neural differentiation, was remarkably up-regulated in the
presence of NA. The expression of left-right-determination factor
(LEFT) A & B as well as PITX1 was down regulated.
* * * * *