U.S. patent application number 14/583838 was filed with the patent office on 2015-05-07 for retinal pigment epithelial cells differentiated from embryonic stem cells with nicotinamide and activin a.
This patent application is currently assigned to HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPMENT LTD.. The applicant listed for this patent is HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPMENT LTD.. Invention is credited to Ruslana ALPER-PINUS, Eyal BANIN, Masha IDELSON, Alex OBOLENSKY, Benjamin Eithan REUBINOFF.
Application Number | 20150125506 14/583838 |
Document ID | / |
Family ID | 39666210 |
Filed Date | 2015-05-07 |
United States Patent
Application |
20150125506 |
Kind Code |
A1 |
IDELSON; Masha ; et
al. |
May 7, 2015 |
RETINAL PIGMENT EPITHELIAL CELLS DIFFERENTIATED FROM EMBRYONIC STEM
CELLS WITH NICOTINAMIDE AND ACTIVIN A
Abstract
The present invention concerns RPE cells obtainable by directed
differentiation from stem cell, particularly, human stem cells. It
has been specifically found that culturing stem cells in the
presence of one or more member of the TGF.beta. superfamily, such
as Activin A) induced directed differentiation into mature and
functional RPE cells. This was evidenced by the expression of
markers specific to mature RPE cells, including MiTF-A, RPE65 or
Bestrophin). In accordance with one particular embodiment, the
cells are a priori cultured with nicotinamide (NA) which was found
to augment the cells' response to the inductive effect of the one
or more member of the TGF.beta. superfamily. The invention also
provides methods of performing the directed differentiation, as
well as methods for use of the resulting RPE cells.
Inventors: |
IDELSON; Masha; (MaAle
Adumim, IL) ; ALPER-PINUS; Ruslana; (Jerusalem,
IL) ; OBOLENSKY; Alex; (Jerusalem, IL) ;
BANIN; Eyal; (Jerusalem, IL) ; REUBINOFF; Benjamin
Eithan; (Moshav Bar-Giora, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HADASIT MEDICAL RESEARCH SERVICES AND DEVELOPMENT LTD. |
JERUSALEM |
|
IL |
|
|
Assignee: |
HADASIT MEDICAL RESEARCH SERVICES
AND DEVELOPMENT LTD.
JERUSALEM
IL
|
Family ID: |
39666210 |
Appl. No.: |
14/583838 |
Filed: |
December 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12450943 |
Oct 19, 2009 |
8956866 |
|
|
PCT/IL2008/000556 |
Apr 27, 2008 |
|
|
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14583838 |
|
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|
60907818 |
Apr 18, 2007 |
|
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Current U.S.
Class: |
424/423 ;
424/93.7 |
Current CPC
Class: |
C12N 2501/999 20130101;
A61P 27/02 20180101; C12N 5/0696 20130101; C12N 2502/13 20130101;
C12N 2501/115 20130101; A61K 35/30 20130101; C12N 2506/02 20130101;
A61P 9/10 20180101; C12N 2500/38 20130101; A61K 35/44 20130101;
C12N 2500/90 20130101; A61K 35/48 20130101; C12N 2501/155 20130101;
A61K 9/0051 20130101; C12N 2533/52 20130101; C12N 5/0621 20130101;
C12N 2501/15 20130101; C12N 2500/99 20130101; C12N 2501/16
20130101 |
Class at
Publication: |
424/423 ;
424/93.7 |
International
Class: |
A61K 35/30 20060101
A61K035/30; C12N 5/079 20060101 C12N005/079; A61K 9/00 20060101
A61K009/00 |
Claims
1. A method of transplanting retinal pigment epithelial (RPE) cells
into a subject's eye, comprising: (a) culturing human stem cells in
a medium comprising a differentiating agent to obtain
differentiating cells; (b) culturing said differentiating cells in
a medium comprising one or more members of the TGF.beta.
superfamily to obtain differentiated RPE cells; (c) harvesting said
differentiated RPE cells; and (d) transplanting said differentiated
RPE cells into the subject's eye, thereby transplanting RPE cells
into the subject's eye.
2. The method of claim 1, wherein said transplanting of said
differentiated RPE cells is effected at the subretinal space of the
eye.
3. The method of claim 1, wherein said RPE cells are transplanted
in a suspension, or as a monolayer of cells immobilized on a matrix
or a substrate.
4. The method of claim 1, wherein said human stem cells comprise
human pluripotent stem cells.
5. The method of claim 4, wherein said human pluripotent stem cells
comprise human embryonic stem cells.
6. The method of claim 1, wherein said differentiating agent
comprises nicotinamide.
7. The method of claim 1, wherein said member of the TGF.beta.
superfamily is TGF.beta.1, TGF.beta.3 or activin A.
8. The method of claim 6, wherein said medium of step (b) comprises
nicotinamide and activin A.
9. A method of treating a retinal disease or disorder in a subject
in need thereof, comprising: (a) culturing human stem cells in a
medium comprising a differentiating agent to obtain differentiating
cells; (b) culturing said differentiating cells in a medium
comprising one or more members of the TGF.beta. superfamily to
obtain differentiated RPE cells; (c) harvesting said differentiated
RPE cells; and (d) transplanting said differentiated RPE cells into
the subject's eye, thereby treating the retinal disease or
disorder.
10. The method of claim 9, wherein said transplanting of said
differentiated RPE cells is effected at the subretinal space of the
eye.
11. The method of claim 9, wherein said RPE cells are transplanted
in a suspension, or as a monolayer of cells immobilized on a matrix
or a substrate.
12. The method of claim 9, wherein said human stem cells comprise
human pluripotent stem cells.
13. The method of claim 12, wherein said human pluripotent stem
cells comprise human embryonic stem cells.
14. The method of claim 9, wherein said differentiating agent
comprises nicotinamide.
15. The method of claim 9, wherein said member of the TGF.beta.
superfamily is TGF.beta.1, TGF.beta.3 or activin A.
16. The method of claim 14, wherein said medium of step (b)
comprises nicotinamide and activin A.
17. The method of claim 9, wherein said retinal disease or disorder
is selected from at least one of retinitis pigmentosa, lebers
congenital amaurosis, hereditary or acquired macular degeneration,
age related macular degeneration (AMD), Best disease, retinal
detachment, gyrate atrophy, choroideremia, pattern dystrophy, RPE
dystrophies, Stargardt disease, RPE and retinal damage due to
damage caused by any one of photic, laser, inflammatory,
infectious, radiation, neovascular or traumatic injury.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 12/450,943 filed on Oct. 19, 2009, which is a National
Phase of PCT Patent Application No. PCT/IL2008/000556 having
International Filing Date of Apr. 27, 2008, which claims the
benefit of priority from U.S. Provisional Patent Application No.
60/907,818, filed on
Apr. 18, 2007. The contents of the above Application are all
incorporated herein by reference.
SEQUENCE LISTING STATEMENT
[0002] The ASCII file, entitled 61243SequenceListing.txt, created
on Dec. 29, 2014, comprising 4,096 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] This invention relates to methods and systems for producing
differentiated retinal pigment epithelial cells (RPE) and to
therapeutic uses of RPE cells obtained thereby.
LIST OF RELATED ART
[0004] The following is a list of references which are considered
to be pertinent for describing the state of the art in the field of
the invention. [0005] (1) Strauss O., The retinal pigment
epithelium in visual function; Physiol. Rev. 85: 845-881, 2005.
[0006] (2) Lund R D. et al., Cell transplantation as a treatment
for retinal disease; Prog Retin Eye Res 20: 415-449, 2001. [0007]
(3) Haruta M., Embryonic stem cells: potential source for ocular
repair; Semin Ophthalmol. 20(1):17-23, 2005. [0008] (4) Haruta M.
et al., In vitro and in vivo characterization of pigment epithelial
cells differentiated from primate embryonic stem cells; Invest
Ophthalmol Vis Sci 45:1020-1024, 2004. [0009] (5) Aoki H. et al.,
Embryonic stem cells that differentiate into RPE cell precursors in
vitro develop into RPE cell monolayers in vivo; Exp Eye Res.
82(2):265-274, 2006. [0010] (6) Klimanskaya I. et al., Derivation
and comparative assessment of retinal pigment epithelium from human
embryonic stem cells using transcriptomics; Cloning Stem Cells
6(3):217-245, 2004. [0011] (7) Lund R D. et al., Human embryonic
stem cell-derived cells rescue visual function in dystrophic RCS
rats; Cloning Stem Cells 8(3):189-199, 2006. [0012] (8) PCT
application publication No. WO 06/070370.
BACKGROUND OF THE INVENTION
[0013] Dysfunction, injury, and loss of retinal pigment epithelium
(RPE) cells are prominent features of certain eye diseases and
disorders, such as age-related macular degeneration (AMD),
hereditary macular degenerations including Best disease (the early
onset form of vitelliform macular dystrophy), and subtypes of
retinitis pigmentosa (RP). A potential treatment for such diseases
is the transplantation of RPE (and photoreceptors) into the retina
of those affected with the diseases. It is believed that
replenishment of RPE cells by their transplantation may delay, halt
or reverse degeneration, improve retinal function and prevent
blindness stemming from such conditions.
[0014] The macula, the central part of the retina, is responsible
for fine visual detail and color perception, and is crucial for
many of our daily visual tasks such as facial recognition and
reading. The macula is often affected as part of the disease
process in widespread retinal degenerations such as retinitis
pigmentosa (RP), as well as in different diseases that more
specifically target the macular region such as age-related macular
degeneration (AMD) and Best disease. In many of these diseases, the
primary dysfunction and failure occurs in the retinal pigment
epithelium (RPE) cells which underlie the photoreceptors.
[0015] The highly specialized RPE cells play a major role in
supporting photoreceptor function: they actively transport
nutrients from the choroidal vessels, participate in the recycling
of vitamin A, which is necessary for the chromophores in the
photoreceptors, and take-up and recycle shed photoreceptor outer
segments as part of the normal renewal process of these
cells.sup.1.
[0016] In subtypes of RP, Best disease, and AMD, failure of the RPE
ultimately leads to visual loss and blindness. Replacement of these
cells is a possible therapeutic intervention.sup.2, but obtaining
such cells from human donors or embryos is difficult. Human
embryonic stem cells (hESCs) may serve as a potential unlimited
donor source for RPE cells, if the means to direct their
differentiation into functional RPE cells can be elucidated.sup.3.
Methods to direct the differentiation of hESCs into cultures highly
enriched for neural precursor cells (NPs) have previously been
described (Reubinoff B E. et al., Neural progenitors from human
embryonic stem cells; Nat Biotechnol 19: 1134-1140, 2001; Itsykson
P. et al., Derivation of neural precursors from human embryonic
stem cells in the presence of noggin; Mol Cell Neurosci.
30(1):24-36, 2005). In addition, the potential of hESCs to give
rise to retinal cells both in vitro and in vivo following
transplantation to the subretinal space in rodents has been shown
(Banin E. et al., Retinal Incorporation and Differentiation of
Neural Precursors Derived from Human Embryonic Stem Cells; Stem
Cells 24(2):246-257, 2006.)
[0017] The potential of mouse and non-human primate ESCs to
differentiate into RPE cells, and to survive and attenuate retinal
degeneration after transplantation, has been demonstrated.sup.4,5.
Spontaneous differentiation of hESCs into RPE cells was shown,
however, the efficiency of the differentiation process was low, a
substantial time of differentiation was required and only a low
(<1%) percentage of clusters containing RPE cells were obtained
after 4-8 weeks of differentiation. Furthermore, while improved
retinal function was observed in RCS rats after sub retinal
transplantation of these RPE cells, function of the transplanted
cells as authentic mature RPE cells was not demonstrated and this
effect could potentially be related to a non-RPE-specific trophic
effect.sup.6,7,9,10.
[0018] It was also recently shown that hESCs may be directed to
reproducibly differentiate into RPE cells, in which directed rather
than spontaneous differentiation of hESCs towards an RPE fate
occurred in the presence of Nicotinamide (NA).sup.8.
SUMMARY OF THE INVENTION
[0019] In accordance with a first aspect, the present invention
provides the use of a member of the transforming growth
factor-.beta. (TGF.beta.) superfamily for the preparation of a
culture system for promoting directed and augmented differentiation
of human stem cells (hSCs) into retinal pigment epithelial (RPE)
cells.
[0020] In accordance with a second aspect, the present invention
provides a method for promoting directed differentiation of hSCs
into RPE fate, the method comprising:
[0021] (a) providing a cell culture comprising hSCs; and
[0022] (b) culturing cells in said cell culture in a culture system
comprising a basic medium supplemented with one or more member of
TGF.beta. superfamily whereby said hSCs are promoted towards
directed differentiation into RPE fate.
[0023] In accordance with a third aspect, there is provided a cell
culture comprising RPE cells obtained by directed differentiation
of hSCs in the presence of one or more member of TGF.beta.
superfamily. Preferably, the RPE cells are terminally
differentiated (mature) RPE cells obtained by the method disclosed
herein. As will be shown herein, such RPE cells exhibit several
characteristic traits that are different from those obtained when
hSCs are spontaneously differentiated into RPE cells. Preferably,
the RPE cells are capable of responding to TGF.beta. signaling
during their differentiation.
[0024] In accordance with a fourth aspect, there is provided a
method of transplanting hSCs-derived RPE cells into a subject's
eye, said RPE cells obtained by directed differentiation of said
hSCs, the method comprises
[0025] (a) providing a cell culture comprising hSCs;
[0026] (b) culturing said cell culture in a culture system
comprising a basic medium supplemented with one or more member of
TGF.beta. superfamily whereby said hSCs are induced to
differentiate into RPE cells;
[0027] (c) harvesting from said cell culture RPE cells; and
[0028] (d) transplanting said RPE cells into said subject's
eye.
[0029] In accordance with a fifth aspect, there is provided a cell
culture system comprising transplantable hSCs-derived RPE cells
obtained by directed differentiation of said hSCs. The transplanted
RPE cells exhibited one or more parameters indicative that said
transplanted cells are functional within said subject eye. The
functionality of the transplanted RPE cells is exhibited by their
ability to uptake shed outer segments of photoreceptors in parallel
to improving retinal function
[0030] The hSCs in the culture system of the methods disclosed
herein are differentiating hSCs, i.e. a population of hSCs
essentially in an undifferentiated state, or wherein at least part
of said cells have been induced to undergo initial stages of
directed differentiation and at times, the majority of said cells
have been induced to undergo initial stages of directed
differentiation. In accordance with one embodiment, the initial
stage of differentiation is achieved by a priori exposing the cells
to NA although initial stages of differentiation will occur also
when the undifferentiated cells are co-exposed to NA and the one or
more member of the TGF.beta. superfamily. Without being bound to
theory, it is postulated that the prior exposure to NA (prior to
incubation with the one or more member of the TGF.beta.
superfamily) primes the cells towards directed differentiation (as
opposed to spontaneous differentiation) into RPE cells with
specific RPE morphology, as will be further discussed below.
[0031] According to a preferred embodiment, the hSCs are human
embryonic stem cells (hESCs).
[0032] According to one embodiment, the culturing of cells in a
medium comprising one or more member of TGF.beta. superfamily is at
least two days after the hSCs have initiated differentiation,
directed differentiation, preferably directed by NA.
[0033] In accordance with a fifth aspect, there is provided a
method of treating or preventing in a subject a retinal disease or
disorder comprising dysfunction, injury, and/or loss of retinal
pigment epithelium, the method comprises intraocular
transplantation to said subject of hSC-derived RPE cells, the RPE
cells obtained by inducing the hSCs towards directed
differentiation. The transplantable RPE cells are preferably
obtained by the method disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0035] In order to understand the invention and to show 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:
[0036] FIGS. 1A-1E: Real-Time PCR, analyzing the expression of RPE
markers in the presence of NA. Differentiation of hESCs was induced
by culturing them as free floating clusters. At 6 weeks of
differentiation, the level of expression of the RPE markers MiTF-A
(FIG. 1A) and RPE65 (FIG. 1B) was significantly enhanced in the
presence of NA. Real-Time PCR analysis at sequential time points
demonstrated the progressive increase in the expression levels of
MiTF-A (FIG. 1C) and RPE65 (FIG. 1D) along time in the presence of
NA. Expression of additional transcripts of RPE markers including
Bestrophin, CRALBP and Mertk were demonstrated by RT-PCR analysis
of plated pigmented clusters (FIG. 1E). +/- indicates presence or
absence, respectively, of reverse transcriptase.
[0037] FIGS. 2A-2F: The RPE differentiation inductive effect of NA
is not dependent on a specific medium composition. Dark field
micrographs of hESC clusters differentiating 12 weeks in KO medium
(FIG. 2A) or in Neurobasal medium supplemented with N2, which is
substituted after 1 week with DMEM/F12 supplemented with B27 (NN
medium) (FIG. 2C). In both media NA augmented the differentiation
towards pigmented cells (FIG. 2B, D) though the size of the
differentiated hESC-clusters and their total number was smaller
with NN medium (white arrows mark pigmented areas within
differentiating clusters). At the RNA level, in both media, NA
supplementation enhanced the level of expression of MiTF-A and
RPE65 (FIGS. 2E, and F; respectively).
[0038] FIGS. 3A-3L: Melanin-expressing cells within free floating
clusters of hESCs are putative RPE cells. Dark field micrograph of
free floating clusters of differentiating hESCs with defined areas
highly enriched for pigmented cells (FIG. 3A). Fluorescence (FIG.
3B) and phase contrast (FIG. 3C) images of pigmented cells
following dissociation and plating that are immunoreactive with
anti Otx2 and MiTF. A dark field micrograph of differentiating
clusters after plating illustrating confined pigmented areas is
shown (FIG. 3D). Phase contrast image of cells within the pigmented
areas having morphological characteristics that are typical of RPE
cells (FIG. 3E). Indirect immunofluorescent stainings showing that
these cells express markers of RPE cells, including MiTF (FIG. 3F),
ZO-1 (FIG. 3G), Bestrophin (FIG. 3H), RPE65 (FIG. 3I), and CRALBP
(FIG. 3J). After dissociation, plating at low density and
cultivation, the pigmented cells lose pigmentation and acquire
fibroid-like morphology (phase contrast image) (FIG. 3K). Following
further prolonged cultivation and proliferation into high density
cultures the cells reacquire the morphology and pigmentation
characteristic of RPE cells (FIG. 3L).
[0039] FIGS. 4A-4E: Activin A induces RPE differentiation. Human
ESCs were allowed to differentiate as free floating clusters for 6
weeks in the absence or presence of activin A which was
supplemented after the first week of differentiation. Dark field
micrographs of the clusters show that activin A significantly
increased the percentage of clusters that included pigmented cells
(FIG. 4A, B) (white arrows mark pigmented areas within
differentiating clusters and the borders of pigmented areas within
some of the clusters are marked by dashed lines). In the presence
of activin A the borders of the pigmented areas are more sharply
demarcated from surrounding non-pigmented areas within the
clusters. Furthermore, the pigmented cells are darker in the
presence of activin A (FIG. 4B). At the RNA level, Real-Time PCR
analysis showed that the expression of RPE65 (FIG. 4D) and
Bestrophin (FIG. 4E) is significantly enhanced in the presence of
activin A. The expression of MiTF-A was not altered by activin A
treatment (FIG. 4C).
[0040] FIGS. 5A-5G: BMPs and TGF.beta.3 have a role in RPE
differentiation. Human ESCs were induced to differentiate as free
floating clusters for 6 weeks. Spontaneous differentiation into
pigmented cells was infrequently observed (FIG. 5A) but was
significantly enhanced when the medium was supplemented with NA
(FIG. 5B, far-left and left dark field images; white arrows mark
pigmented areas within differentiating clusters). Supplementation
of the medium by noggin blocked the differentiation into pigmented
cells both in the absence (FIG. 5D) and in the presence of NA (FIG.
5C). At the RNA level, Real-Time PCR analysis showed that noggin
reduced the expression levels of MiTF-A both in the presence and
absence of NA (FIG. 5E). When TGF.beta.3 was added to the culture
medium during differentiation of hESC clusters in the presence of
NA, it significantly augmented the expression levels of MiTF-A
(FIG. 5F) but not of RPE65 (FIG. 5G).
[0041] FIGS. 6A-6P: Survival and integration of transplanted
hESC-derived RPE cells in rat eyes. Following intraocular
transplantation of hESC-derived RPE cells, the pigmented cells
could be readily identified in vivo in the eyes of albino rats
(FIGS. 6A, 6B). Following enucleation of the eye (FIG. 6B) and
removal of the cornea and lens, the main graft as well as
additional dispersed pigmented spots could be seen (FIG. 6C). In
histological sections, grafts that included dark pigmented cells
which also co-express GFP could be identified (FIG. 6D-6G),
attesting to the fact that the cells were hESC-derived.
Transplanted cells could be found in the intravitreal area, between
the retina and lens (FIG. 6H), in the retina (occasionally
protruding into the vitreous along the tract of injection) (FIG.
6I), and also in the subretinal space (FIGS. 6I, 6O, 6P).
Transplanted hESC-derived RPE cells (pigmented cells marked with
arrows) integrated into the RPE layer of albino rats (FIG. 6J).
Pigmented cells were not observed in the RPE layer of control
non-transplanted eyes. Within grafts, immunostaining with ZO-1
(FIG. 6K-6N) showed that there were tight junctions between
transplanted GFP+hESC-derived cells. Such junctions are
characteristic of RPE cells. Following transplantation into the
subretinal space of RCS rats with RPE dysfunction and retinal
degeneration, relative preservation of the photoreceptor layer
could be seen in proximity to the graft (FIG. 6O; the area within
the rectangle is marked by an asterisk, and enlarged in FIG. 6P),
as compared to areas distant from the graft (marked by arrows).
Note the large transplanted hESC-derived RPE cells with polygonal
shape and cobblestone-like appearance (FIG. 6P) (asterisk). In all
cases shown here, RPE cells were derived from hESCs without
presence of Activin-A.
[0042] FIG. 7: Electroretinographic recordings show that
transplantation of hESC-derived RPE cells provides rescue of
retinal function in the eyes of dystrophic RCS rats. Full field ERG
responses are higher in RCS rat eyes following transplantation of
RPE cells derived from hESC compared to fellow non-transplanted
control eyes (n=11 rats). RPE cells used in these experiments were
derived without addition of Activin A to the culture medium. b-wave
amplitudes of the dark-adapted mixed cone-rod responses to four
stimuli of increasing intensity are shown.
[0043] FIGS. 8A-8I: Analysis of morphology and marker expression
showing the effect of NA in inducing the development of pigmented
cells from hESCs. Dark field micrographs showing the progressive
appearance of pigmented cells during culturing of hESC-derived
clusters for 4 weeks (FIG. 8A, 8B), 6 weeks (FIGS. 8C, 8D) and 8
weeks (FIG. 8E, 8F) in the presence (FIGS. 8A, 8C, and 8E) or
absence of NA (FIG. 8B, 8D or 8F) (white arrows mark pigmented
areas within differentiating clusters). Histogram presentation of
the percentage of clusters containing pigmented areas at different
time points during culture in medium supplemented with NA (bars
with bold line) and in control cultures (bars with fine line) (FIG.
8G). Histogram presentation of the percentage of pigmented cells
(FIG. 8H). and the cells that are immunoreactive with anti-MiTF
(FIG. 8I), an early RPE marker, during 8 weeks of culture with NA
supplementation Scale bars: (A) 200 .mu.m; *p<0.05;
**p<0.001.
[0044] FIGS. 9A-9S: Real-time PCR, immunostaining and flow
cytometry analysis showing the progression of RPE development along
time in hESC differentiating clusters. (FIG. 9A-9L) Real-time PCR,
analyzing the timing of the expression of key genes in RPE
development in clusters cultured in the presence (bars with bold
line) or absence (bars with fine line) of NA. The progressive
expression of the following markers was analyzed at sequential time
points during 8 weeks differentiation of hESC-derived clusters: the
hESC-specific marker, Oct4 (FIG. 9A); the early neural markers,
Otx2 (FIG. 9B), Musashi (FIG. 9C) and Pax6 (FIG. 9D); the retinal
progenitor markers, Six3 (FIG. 9E), Rx1 (FIG. 9F), and Chx10 (FIG.
9G); the RPE markers, MiTF-A (FIG. 9H), RPE65 (FIG. 9I) and
bestrophin (FIG. 9J); the photoreceptor progenitor marker, Crx
(FIG. 9K); the melanocyte developmental marker, Sox10 (bars with
horizontal stripes, FIG. 9L) (The M51 melanoma cell line is used as
a control). FACS analysis demonstrating the progressive expression
of the hESC-specific marker, TRA-1-60 (FIG. 9M), and the neural
progenitor marker, PSA-NCAM (FIG. 9O), in clusters differentiating
for 8 weeks in the presence bars with (bold line) or absence (bars
with fine line) of NA. Indirect immunofluorescence analysis of the
percentage of cells expressing the early neural markers: PSA-NCAM
(bars with bold line), nestin (bars with horizontal stripes),
Musashi (bars with fine line), Pax6 (bars with vertical stripes),
within the clusters differentiating for 2 and 4 weeks in the
presence of NA (FIG. 9N). Immunofluorescence images, demonstrating
the cells expressing these markers, PSA-NCAM (FIG. 9P), nestin
(FIG. 9Q), musashi (FIG. 9R), Pax6 (FIG. 9S).
[0045] FIG. 10A-10J: Analysis of morphology, marker expression and
function showing that the pigment-expressing cells within
free-floating clusters of hESCs are putative RPE cells. Phalloidine
staining showing distribution of F-actin within the hESC-derive
pigmented progeny which is characteristic of RPE (FIG. 10A); After
dissociation, plating at low density and cultivation, the pigmented
cells lost pigmentation and acquired fibroid-like morphology (phase
contrast image, 1 week of culture) (FIG. 10B). Following further
prolonged cultivation and proliferation into high density cultures
the cells reacquired the morphology and pigmentation
characteristics of RPE cells (1.5 month of culture) (FIG. 10C).
Electron microscopic analysis of hESC-derived RPE cells showing
features characteristic of RPE: microvilli (FIG. 10D), a basal
membrane (FIG. 10E), melanin granules (FIG. 10D), tight junctions
(FIG. 10F). Phase contrast FIG. 10G and fluorescent images (FIG.
10H-J) showing phagocytosis of green fluorescent latex beads (Wight
arrowheads) by the hESC-derived pigment cells; the cell membranes
were stained with red fluorescent die PKH (grey color). The three
confocal fluorescent images demonstrated serial z axis slices (FIG.
10H-J).
[0046] FIGS. 11A-11P: Analysis of morphology and gene expression
showing that factors from the TGF.beta. family promote
differentiation towards RPE fate. Dark field micrographs of
hESC-derived clusters differentiating for 4 weeks showed the
appearance of pigmented cells at this early stage in the presence
of activin (FIG. 11A). as well as the increase in number of
pigmented clusters differentiating in the presence of activin A and
NA (FIG. 11C) as opposed to NA only (FIG. 11B). Similar to activin
A, supplementation with TGF.beta.1 also increases the appearance of
pigmented clusters (FIG. 11D). In contrast, the application of the
inhibitor of activin signaling pathway, SB431542, together with
activin A and NA reduced the effect of activin A on the appearance
of pigmented clusters (FIG. 11E). The development of pigmented
clusters was also abolished by culturing the cells in the presence
of FGF.beta. together with NA (FIG. 11F). Expression of transcripts
of activin receptors and activin A was demonstrated by RT-PCR
analysis of 2 weeks old clusters cultured in the presence or
absence of NA and undifferentiated hESCs as controls (FIG. 11G).
Histogram analysis of the percentage of clusters containing
pigmented areas at 4 weeks following culture in the presence of NA,
NA+ActA, NA+SB431542, NA+ActA+SB431542, NA+TGF.beta.1 (FIG. 11H).
Histogram analysis of the percentage of pigmented cells after 4
weeks of culture with NA (bars with bold line) or activin A and NA
(bars with diagonal stripes) supplementation (FIG. 11I). Histogram
analysis of the percentage of pigmented cells (FIG. 11J) and the
level of expression of transcripts of the RPE markers, Bestrophin
(FIG. 11K) and RPE65 (FIG. 11L) at different concentrations of
activin A, that 140 ng/ml is optimal for RPE induction. Real-time
PCR time-course analysis of the effect of activin A on the
expression levels of retinal and RPE genes, Bestrophin (FIG. 11M),
MiTF-total (FIG. 11N), Rx1 (FIG. 11O) and Chx10 (FIG. 11P), in
hESCs differentiating in the presence of NA with (bars with
diagonal stripes) or without (bars with bold line) activin A
supplementation **p<0.005. (white arrows mark pigmented areas
within differentiating clusters).
[0047] FIG. 12A-12E: RPE cells derived from hESCs treated by NA and
Activin A survive following sub-retinal transplantation in
dystrophic RCS rat eyes. Clusters of pigmented cells could be
readily identified in-vivo in the eyes of RCS rats using fundus
imaging systems (FIGS. 12A-12C); Fundus photo (FIG. 12A) and
red-free photo (FIG. 12B) which showed the subretinal location of
the grafts (note retinal vessels coursing over the pigmented
areas). The hESC-derived, GFP-expressing cells can be seen to emit
fluorescence when fluorescein excitation and emission filters were
used (FIG. 12C). In eye cup preparations imaged ex-vivo in a
fluorescence microscope (FIGS. 12D-12E), large clusters of
subretinal GFP-positive cells can be seen (FIG. 12D) as well as
multiple, dispersed smaller clusters (FIG. 12E).
[0048] FIG. 13A-13F: Histologic appearance of sub-retinal
hESC-derived, Activin-A-treated RPE cell grafts in RCS rat eyes.
Histologic sections stained by hemotoxylin and eosin (FIGS. 13A,
and 1B) showed the subretinal and occasionally intra-retinal
location of transplanted hESC-derived pigmented cells, which
appeared in clusters or as isolated cells (arrows). Immunostaining
with GFP (FIGS. 13C-13F) confirmed that the cells are indeed
hESC-derived. Grafts were often quite large and dispersed (FIGS.
13C, 13E), and pigmented cells co-expressing GFP can be clearly
seen (FIGS. 13D, 13F). Note GFP-positive pigmented cells
integrating within the host RPE layer (FIG. 13D, arrow).
[0049] FIG. 14A-14O: Transplanted hESC-derived pigmented cells
express markers of mature RPE. Immunostaining revealed that large
numbers of transplanted cells within grafts express proteins which
are characteristic of mature RPE cells, including the RPE-specific
markers RPE65 (FIGS. 14A-14E) and Bestrophin (FIGS. 14F-14J) as
well as the tight-junction marker ZO-1 (FIGS. 14K-14O). FIGS. 14A,
14F and 14K show low-magnification fluorescent image of grafts
co-expressing GFP and the relevant marker. High magnification
confocal images in each row show pigment (by Nomarski optics) as
well as co-expression of GFP and the different markers at the
single-cell level. These series confirm that the cells are indeed
hESC-derived and that they express markers of mature RPE in-vivo.
In FIG. 14M, note that the host RPE stains for ZO-1 (dashed arrow)
while it is GFP-negative in FIGS. 14N, 14O (corresponding area is
dark) as opposed to the ZO-1 positive hESC-derived cells (full
arrow in FIG. 14M) which do co-express GFP (FIGS. 14N, 14O).
[0050] FIG. 15A-15C: Transplanted hESC-derived, Activin-A treated
RPE cells provide functional rescue in the RCS rat retinal
degeneration model. Full field ERG responses recorded at the age of
8 weeks were higher in RCS rat eyes following transplantation of
activin-treated RPE cells derived from hESC as compared to fellow
non-transplanted control eyes as well as compared to eyes in which
subretinal injection of medium alone was performed. Representative
ERG responses to a series of white flashes of increasing intensity
in the dark-adapted state are shown in a transplanted eye
[0051] (FIG. 15A) versus its fellow control eye (FIG. 15B). FIG.
15C shows the marked difference in mean amplitudes between
transplanted eyes and the different groups of control eyes
(--.diamond-solid.-- injected eye (n=13); --.box-solid.-- non
injected eye (n=13); -- -- medium non-injected eyes (n=5);
--.tangle-solidup.-- medium injected eye (n=5)). As shown, there is
a trend towards better preservation of retinal function following
transplantation of activin-A treated RPE cells (shown here) as
compared to the rescue effect achieved following transplantation of
RPE cells derived without activin-A (FIG. 7).
[0052] FIG. 16A-16D: Transplanted hESC-derived, Activin-A treated
RPE cells provide structural rescue in the RCS rat retinal
degeneration model. The effects of transplanted hESC-derived,
activin-treated RPE cells on the degenerating host retina were
examined and quantified using high resolution microscopic images of
hematoxylin and eosin stained sections. Relative preservation of
the outer nuclear (photoreceptor) layer (ONL) and of the inner and
outer photoreceptor segments (IS+OS) was observed in proximity to
sub-retinal RPE grafts as compared with areas distant from the
grafts (two examples shown in FIG. 16A, 16B). Inserts in FIG. 16A
demonstrate this difference (rescued retina with relatively thick
ONL shown in right insert in proximity to graft; severe thinning of
the ONL is seen in left insert, distant from the graft). Total
retinal thickness (FIG. 16C) as well as ONL and IS+OS thickness
(FIG. 16D) were significantly increased in vicinity to hESC-derived
RPE grafts (black bars, mean.+-.SEM, n=7) as compared to areas
distant from grafts (gray bars). This type of structural rescue was
observed only in proximity to sub-retinal and deep intra-retinal
grafts, and not when grafts were exclusively intra-vitreal (not
shown). For details of quantification technique, please see
methods.
[0053] FIG. 17A-17E: Transplanted hESC-derived, Activin-A treated
RPE cells uptake rhodopsin in-vivo. Confocal images of subretinal
transplanted RPE cells show the co-localization of pigment, GFP,
RPE65 and rhodopsin within the same single cells. The native RPE
cells of the RCS rat express RPE65 (FIG. 17C, arrow) but do not
express GFP (FIG. 17D, arrow) and contain minimal amounts of
rhodopsin (FIGS. 17B, 17E).
DETAILED DESCRIPTION OF THE INVENTION
[0054] The present disclosure provides the use of one or more
members of the transforming growth factor-.beta. (TGF.beta.)
superfamily for the preparation of a culture system for promoting
differentiation of human stem cells (hSCs), preferably human
embryonic stem cells (hESCs) into retinal pigment epithelial (RPE)
cells. It should be noted that in addition to the specified uses
discussed in detailed herein, also encompassed within the present
disclosure are RPE cells obtained by directed differentiation of
hSCs in the presence of one or more the TGF.beta. superfamily; as
well as a method for promoting directed differentiation of hSCs
into RPE fate, as well as methods for growing and maintaining such
hSCs-derived RPE cells and methods making use of such hSCs-derived
RPE cells. In accordance with some preferred embodiments, the RPE
cells obtained according to the teaching herein are mature (in
other words, terminally differentiated) and functional RPE cells,
as will be further discussed and explained below.
[0055] The present disclosure broadly concerns the use of one or
more members of the TGF.beta. superfamily of growth factors in
promoting/inducing/augmenting the directed differentiation of hSCs
into RPE cells, preferably mature RPE cells.
[0056] In the following description and claims use will be made, at
times, with a variety of terms, and the meaning of such terms as
they should be construed in accordance with the present teaching is
as follows:
GLOSSARY
[0057] "Transforming growth factor-.beta. (TGF.beta.) superfamily
growth factor", as used herein, denotes any member of the TGF.beta.
superfamily of growth factors, such as transforming growth
factor-.beta. proteins, including the TGF.beta.1, TGF.beta.2, and
TGF.beta.3 subtypes, as well as homologous ligands including
activin (e.g., activin A, activin B, and activin AB), nodal,
anti-mullerian hormone (AMH), some bone morphogenetic proteins
(BMP), e.g. BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, and growth and
differentiation factors (GDF).
[0058] "Human stem cells" or "hSCs", as used herein, refers to
cells of human origin which under suitable conditions are capable
of differentiating into other cell types having a particular,
specialized function while under other suitable conditions are
capable of self renewing and remaining in an undifferentiated
pluripotential state as detailed below.
[0059] A "cell" as used herein 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. The hSCs cells are
preferably hematopoietic or mesenchymal stem cells obtained from
bone marrow tissue of an individual at any age or from cord blood
or tissue of a newborn individual, neural stem cells obtained from
fetal, any age post birth, or cadaver brain, embryonic stem (ES)
cells obtained from the embryonic tissue formed after fertilization
(e.g., blastomere, blastocyst), or embryonic germ (EG) cells
obtained from the genital tissue of a fetus any time during
gestation, preferably before 10 weeks of gestation. The term cell
may denote a single cell or a cluster of cells.
[0060] "Embryonic stem cell" and "Pluripotent embryonic stem cell",
as used herein, refer to a cell which can give rise to any
differentiated cell types in an embryo or an adult, including the
germ cells (sperm and eggs).
[0061] "Cell culture" or "Cultured cell", as used herein, refer to
cells or tissues that are cultured, cultivated or grown in an
artificial, in vitro environment.
[0062] "Undifferentiated pluripotential hSCs", "Pluripotent hSCs"
as used herein, refer to precursor cells of human source that have
the ability to form any adult cell. Such cells are true cell lines
in that they: (i) are capable of extensive 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.
hESCs are derived from fertilized embryos that are less than one
week old (in the cleavage or blastocyte stage) or produced by
artificial means (such as by nuclear transfer) that have equivalent
characteristics. Other pluripotent hSCs include, without being
limited thereto, multipotent adult progenitor cells (MAPs), induced
pluripotent stem cells (iPS cells) and amniotic fluid stem
cells.
[0063] "Undifferentiated", as used herein, refers to cultured cells
when a substantial proportion (at least 20%, and possibly over 50%
or 80%) of the cells and their derivatives in the population
display characteristic markers and morphological characteristics of
undifferentiated cells, distinguishing them from differentiated
cells of embryo or adult origin. Cells are recognized as
proliferating in an undifferentiated state when they go through at
least 1 population doubling during a cultivation period of at least
3 weeks, while retaining at least about 50%, or the same proportion
of cells bearing characteristic markers or morphological
characteristics of undifferentiated cells after said cultivation
period.
[0064] "Cell suspension" or "freely floating cells" as used herein,
refers to a culture of cells in which the majority of the cells
freely float in the medium, typically a culture medium (system),
and the cells floating as single cells, as cell clusters and/or as
cell aggregates. In other words, the cells survive and propagate in
the medium without being attached to a substrate.
[0065] "Culture system", as used herein, refers to a culture system
suitable for the propagation of SCs. The term denotes a combination
of elements, at minimum including a basic medium (a cell culture
medium usually comprising a defined base solution, which includes
salts, sugars and amino acids) and the one or more member of a the
transforming growth factor-.beta. (TGF.beta.) superfamily of growth
factors. The culture system in accordance with the invention may
further comprise other elements such as, without being limited
thereto, a serum or serum replacement, a culture (nutrient) medium
and other exogenously added factors, which together provide
suitable conditions that support SC growth as well as other
components typically used in cell culture systems. The above
elements may be collectively classified as soluble elements.
However, in the context of the present invention, the elements may
also be associated to a carrier, i.e. non-soluble elements. The
association may be by chemical or physical attachment/binding. For
example, the element may be immobilized onto a matrix (e.g.
extracellular matrix), presented by cells added to the system or
bound to biodegradable material. Further, the element may be
released from a carrier, the carrier may be a cell or a vesicle
encapsulating or embedding the element. Thus, in the following
text, elements supplementing the basic media to form the culture
system comprise both soluble and non-soluble elements.
[0066] "Differentiation", as used herein, refers to the process of
switching the state of a cell from one cell type to another, and
more specifically in the context of the present disclosure
indicates the process of a human stem cell acquiring the cell type
of a retinal pigment epithelial (RPE) cell with at least one
characteristic feature indicative that said RPE cell is a mature
(terminally differentiated) cell. As used herein, the term "cell
type" refers to a distinct morphological or functional form of a
cell.
[0067] "Differentiating hSCs" as used herein, refer to
undifferentiated hSCs which under suitable conditions are capable
of differentiating in an augmented, directed fashion into a
predetermined fate; the term also referring to a population of hSCs
in which at least part thereof has already been induced to undergo
at least initial differentiation, namely directed differentiation
or combination of same.
[0068] "Prime", "augment" "promote" or "direct", as used herein
interchangeably unless the context dictates otherwise, refer to
initiating non-spontaneous differentiation of stem cells into RPE
cells.
[0069] "Differentiation inducer" or "differentiation promoter",
"differentiation priming agent" or "differentiation promoting
factor" as interchangeably used herein denotes any agent which is
capable of priming, augmenting, promoting or directing
differentiation of pluripotent SCs into a somatic cell, preferably,
into RPE cells.
[0070] "Retinal pigment epithelial cells", "RPE cells", "RPEs",
which may be used interchangeably as the context allows, mean cells
of a cell type functionally similar to that of native RPE cells
which form the pigmented cell layer of the retina (e.g. upon
transplantation within an eye, they exhibit functional activities
similar to those of native RPE cells). Thus, the terms "retinal
pigment epithelial cells", "RPE cells", or "RPEs" may be used to
refer to both native RPE cells of the pigmented layer of the retina
and RPE cells directly differentiated from hSCs, in accordance with
the present disclosure.
[0071] The term "hSC-derived RPE cells" is used herein to denote
RPE cells that are obtained by directed differentiation from hSCs.
In accordance with a preferred embodiment, the hSC-derived RPE
cells are mature (terminally differentiated) and functional RPE
cells as exhibited by parameters defined hereinbelow. The term
"directed differentiation" is used interchangeably with the term
"RPE induced differentiation" and is to be understood as meaning
the process of manipulating hSCs under culture conditions which
induce/promote differentiation into RPE cell type only.
[0072] "Functional RPE cells" is used herein to denote cells
obtained by directed differentiation of hSCs in the presence of one
or more members of the TGF.beta. superfamily, the RPE cells
exhibiting at least one of the following characteristics: [0073]
during differentiation, the cultured cells respond to TGF.beta.
signaling; [0074] the RPE cells are mature, terminally
differentiated cells as exhibited by the expression of markers
indicative of terminal differentiation, e.g. bestrophin or RPE65 as
well or alternatively, by their lack of potency to proliferate in
vivo. [0075] following transplantation (i.e. in situ), the RPE
cells exhibit trophic effect supporting photoreceptors adjacent to
RPE cells; [0076] further, in situ the RPE cells are capable of
functioning with phagocytosis of shed photoreceptor outer segments
as part of the normal renewal process of these photoreceptors.
[0077] Thus, the RPE cells in accordance with the present invention
are especially suitable for regeneration of host RPE thereby
providing improved vision following transplantation therewith into
a subject's eye.
[0078] "Similar", when used in the context of differentiated RPE
cells, means that the differentiated RPE cells share one or more
distinct morphological or functional features with native RPE
cells. For example, sufficient similarity might be indicated by,
for example, determining that the differentiated cell expresses one
or more markers of naturally occurring RPE cells, such as MiTF,
ZO-1, Bestrophin, RPE65, Otx2, Mertk, and CRALBP; or that the cell
manifests one or more physical morphological features of RPE cells,
such as typical F-actin distribution within the cells, pigmentation
by pigmented granules, polygonal (e.g., hexagonal) shape, a
cobblestone-like appearance and ultrastructural features of RPE as
demonstrated by electron microscopy. In addition, may include any
one of the functions listed above, e.g., trophic effect supporting
photoreceptors adjacent to RPE cells; functionality with
phagocytosis of shed photoreceptor outer segments that harbor
rhodopsin or lack of potency to proliferate in vivo.
[0079] "Large scale", as used herein with regard to cell
cultivation and expansion, refers to the production of RPE cells
under conditions which permit at least the doubling of cells in the
cell culture after 4 weeks, the cell population after the 4 weeks
consisting essentially of RPE cells.
[0080] "Cell marker", as used herein, refers to any phenotypic
feature of a cell that can be used to characterize it or
discriminate it from other cell types. A marker may be a protein
(including secreted, cell surface, or internal proteins; either
synthesized or taken up by the cell); a nucleic acid (such as an
mRNA, or enzymatically active nucleic acid molecule) or a
polysaccharide. Included are determinants of any such cell
components that are detectable by antibody, lectin, probe or
nucleic acid amplification reaction that are specific for the
marker of the cell type of interest. The markers can also be
identified by a biochemical or enzyme assay or biological response
that depends on the function of the gene product. Associated with
each marker is the gene that encodes the transcript, and the events
that lead to marker expression. A marker is said to be
preferentially expressed in an undifferentiated or differentiated
cell population, if it is expressed at a level that is at least 50%
higher (in terms of total gene product measured in an antibody or
PCR assay) or 30% more frequently (in terms of positive cells in
the population) than an acceptable control such as actin or
glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Markers that are
expressed 2, 10, 100, or 10,000 times higher or more frequently are
increasingly more preferred.
[0081] The present disclosure makes use of hSCs from which RPE are
derived, due to inductive and directed differentiation of the hSCs
in the presence of a unique culture system applied to the hSCs in
suspension.
[0082] Non-limiting examples of hSCs are neural stem cells obtained
from the fetus, or at any age post birth or from cadaver,
hematopoietic stem cells obtained from bone marrow tissue of a
human individual at any age or from cord blood of a newborn
individual, mesenchymal stem cells, amniotic fluid stem cells,
embryonic stem (ES) cells obtained from the embryonic tissue formed
after fertilization (e.g., from a single blastomere, or from
blastocyst), embryonic germ (EG) cells obtained from the genital
tissue of a fetus any time during gestation, preferably before 10
weeks of gestation, induced pluripotent stem cells, or stem cells
obtained from the gonads of human individual at any age. Preferred
human stem cells according to the present invention are human
embryonic stem cells (hESC).
[0083] hSCs can be obtained using well-known cell-culture methods.
For example, hESC can be isolated from single blastomeres of the
cleavage or morula stage human embryo, from cleavage stage and
morula human embryos and human blastocysts. Human embryos may be
obtained from in vivo preimplantation embryos or more typically
from in vitro fertilized (IVF) embryos. Alternatively,
non-fertilized human oocyte can be parthenogenetically activated to
cleave and develop to the blastocyst stage. In addition a single
cell human embryo can be expanded to the blastocyst stage. For the
isolation of hESCs from a blastocyst, the zona pellucida is removed
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 mechanical dissociation or by
enzymatic digestion 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 ESCs are then
routinely split every 1-2 weeks. For further details on methods of
preparation of hESCs 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]; Bongso et al. [Hum Reprod 4:
706, 1989].
[0084] Commercially available hSCs can be also used in accordance
with the invention. HSCs can be purchased from the NIH human
embryonic stem cells registry. Non-limiting examples of
commercially available embryonic stem cell lines are BG01, BG02,
BG03, BG04, CY12, CY30, CY92, CY10, TE03 and TE32.
[0085] Potential applications of hESC and cells derived from them
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, high through put screening of molecules for their toxic,
regenerative, protective or any other effect, 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].
[0086] There are, however, significant hurdles to the practical
exploitation of hESC.
[0087] Two such hurdles include: maintaining hESC in an
undifferentiated, pluripotential state without spontaneous
differentiation; and directing the differentiation of hESC into
specific types of somatic cells. Several culture systems have been
described for maintaining and propagating stem cells, and
particularly hESC, in an undifferentiated state.sup.8.
[0088] Because of the potential of differentiated cells derived
from stem cells in countless therapeutic applications, directing or
promoting the differentiation of stem cells in culture toward a
specific somatic cell fate is of great interest.
[0089] In certain eye diseases and disorders of, e.g., the retina
and the macula, failure of RPE cells ultimately leads to visual
loss and even blindness. Transplantation of RPE cells to replace
and support the failing host RPE has been suggested as a possible
therapeutic intervention, but obtaining such cells from human
donors or embryos is difficult. hSCs thus may serve as a potential
unlimited donor source for RPE cells, if the means to direct their
differentiation into functional RPE cells can be elucidated.
[0090] It has now been surprisingly found that contacting hSCs with
a member of the TGF.beta. superfamily of growth factors strongly
promotes differentiation of hSCs towards an RPE fate. In other
words, these growth factors have an inducting effect on the hSCs.
Thus, the use of member/members of the transforming growth
factor-.beta. (TGF.beta.) superfamily for the preparation of a
culture system for inducing differentiation of human stem cells
(hSCs) into retinal pigment epithelial (RPE) cells, has thus been
envisaged.
[0091] While many members of the TGF.beta. superfamily of growth
factors are known (some non-limiting examples being listed above),
according to a preferred embodiment, the member of the TGF.beta.
superfamily is preferably the TGF.beta.1, TGF.beta.3 growth factors
or activin A or a combination of same.
[0092] It was previously found that nicotinamide (NA) in a cell
culture has an inhibitory effect on differentiation of stem cells
into extraembryonic cells, and further that NA promotes somatic
differentiation toward neural and further toward an RPE cell-like
fate.sup.8. NA, also known as "niacinamide", is the amide
derivative form of Vitamin B3 (niacin) which is thought to preserve
and improve beta cell function. NA has the chemical formula
C.sub.6H.sub.6N.sub.2O. NA is essential for growth and the
conversion of foods to energy, and it has been used in arthritis
treatment and diabetes treatment and prevention.
##STR00001##
[0093] In the context of the present disclosure, the term NA also
denotes derivatives of NA.
[0094] The term "derivative of nicotinamide (NA)" as used herein
denotes a compound which is a chemically modified derivative of the
natural NA. The chemical modification may include, for example, a
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 derivatives of NA are described, some also
in connection with an inhibitory activity of the PDE4 enzyme
(WO03/068233; WO02/060875; GB2327675A), or as VEGF-receptor
tyrosine kinase inhibitors (WO01/55114). For example, the process
of preparing 4-aryl-nicotinamide derivatives (WO05/014549).
[0095] In connection with the above it has now been surprisingly
found that when hSCs are differentiating in the presence of NA,
their properties are altered and accordingly they gain the
capability to respond to an inductive effect of one or more members
of the TGF.beta. superfamily which directs their differentiation
towards RPE fate, preferably mature and functional RPE cells. Hence
the RPE differentiation-inductive effect of NA may be significantly
enhanced when thereafter exposing the hSCs to the one or more
member of the TGF.beta. superfamily of growth factors in
combination with the pre-exposure of the cells in the culture to
NA.
[0096] Thus, according to one embodiment the method comprises
treating the cells with the one or more member of the TGF.beta.
superfamily of growth factors is used in combination with prior
exposure to NA. Combination may be for the preparation of a culture
system comprising both TGF.beta. and NA; as well as for the
preparation of a culture system comprising only the one or more
member of the TGF.beta. superfamily, to be used for
inducing/promoting differentiation and/or further differentiation
of hSCs which have already been exposed to NA. Without being bound
to theory, it is believed that NA acts as a differentiation
inducer/promoter and that similarly, the one or more members of the
TGF.beta. superfamily act as an RPE differentiation promoting
factor. In addition, while not being bound by theory, it is
believed that the prior exposure of the hSCs to NA provides the
cells with properties that enable their response to the RPE
differentiation promoting effect of the one or more members of the
TGF.beta. superfamily.
[0097] Thus, in accordance with an embodiment of the invention, the
hSCs are first cultured in a culture system comprising a basic
medium supplemented with NA for at least several hours, preferably
at least a day and more preferably, at least two days prior to
culturing the cells in that cell culture in a basic medium (the
same or different) supplemented with the one or more member of
TGF.beta. superfamily.
[0098] In accordance with another embodiment, the undifferentitated
hSCs are cultured in a culture system comprising a basic medium
supplemented with NA and the one or more member of TGF.beta.
superfamily.
[0099] It is noted that various basic mediums are known in the art
for use in cell cultures and preferably for use in SC cultures. A
non-limiting list of basic mediums which can be used in accordance
with the present disclosure comprises Neurobasal.TM.
(CAT#21103-049, Gibco 1998/1999), KO-DMEM (CAT#10829-018, Gibco
1998/1999), DMEM (CAT#41965-039, Gibco 2004), DMEM/F12
(CAT#21331-020, Gibco 2004), Cellgro.TM. Stem Cell Growth Medium
(CAT#2001, CellGenix 2005), or X-Vivo.TM. (CAT#04-380Q, LONZA
2007).
[0100] The present disclosure also provides a method for inducing
directed differentiation of hSCs into RPE fate, the method
comprising: [0101] (a) providing cell culture comprising hSCs;
[0102] (b) culturing cells in said cell culture in a culture system
comprising a basic medium supplemented with one or more members of
the TGF.beta. superfamily whereby directed differentiation of the
hSCs into RPE fate is promoted.
[0103] Differentiation may occur within free floating clusters of
hSCs or adherent cultures. Somatic differentiation within adherent
cultures was described [U.S. Pat. No. 7,112,437]. Such adherent
cultures may thus serve as a basis for inducing RPE differentiation
by a culture system supplemented with at least one or more members
of the TGF.beta. superfamily of growth factors.
[0104] The cells in the cell culture may be a population of
undifferentiated hSCs or a population of cells in which at least
part of the hSCs have initiated differentiation. The initial
differentiation is a directed differentiation. Thus, in the context
of the disclosure, the cells provided in the method are at times
referred to as differentiating cells.
[0105] As already indicated above, the basic medium may be
supplemented by the introduction thereto of soluble elements as
well as by non-souble elements. With respect to the supplementation
with one or more members of the TGF.beta. superfamily of growth
factors, the member may be presented in soluble form or affixed or
associated to a matrix or cell added to the culture system or the
element may be bound or complexed to other substances. The member
may also be secreted to the culture system from cells present in
the latter.
[0106] The hSCs may be provided in undifferentiated state as well
as after being exposed to a differentiation promoting factor
(differentiation priming agent), such as NA. Undifferentiated hSCs
may be obtained from various culture systems in which hSCs may be
maintained in an undifferentiated pluripotent state. For example,
the cells may be cultivated in a feeder-free adherent or suspension
system (WO06/070370) or on feeder cells. Commonly used feeder cells
include a primary mouse embryonic fibroblast (PMEF), a mouse
embryonic fibroblast (MEF), a murine fetal fibroblast (MFF), a
human embryonic fibroblast (HEF), a human fibroblast obtained from
the differentiation of human embryonic stem cells, a human fetal
muscle cell (HFM), a human fetal skin cell (HFS), a human adult
skin cell, a human foreskin fibroblast (HFF), a human cell obtained
from the umbilical cord or placenta, a human adult fallopian tubal
epithelial cell (HAFT) and human marrow stromal cells (hMSCs). The
clusters of hSCs may be obtained from an adherent cell culture by
dissociation of the cells from the feeder layer or extracellular
matrix to form a suspension of cells. The suspension of cells may
comprise the free floating clusters or an essentially single cell
suspension from which clusters of cells are outgrown to form the
cell clusters.
[0107] In accordance with a preferred embodiment, the cell culture
comprises cell suspension, preferably free floating clusters in a
suspension culture, i.e. aggregates of cells derived from human
embryonic stem cells (hESCs). Sources of free floating stem cells
were previously described in WO 06/070370, which is herein
incorporated by reference in its entirety.
[0108] The culturing step in accordance with the present disclosure
may comprise cultivation of the cells in the cell culture with one
or more different culture systems, at least one of the culture
systems comprising the one or more member of the TGF.beta.
superfamily.
[0109] In accordance with one embodiment of the present disclosure,
the cells in the culture are cultivated in a culture system
comprising a basic medium supplemented with NA, in addition to said
one or more member of TGF.beta. superfamily of growth factors.
[0110] In accordance with another embodiment, the cells are firstly
cultured in a culture system comprising a basic medium and NA, the
cells being undifferentiated hSCs, and preferably after hSCs
differentiation is induced (i.e. after a predetermined time period
or after confirming cell differentiation by techniques available in
the art) the cells in the cell culture are cultured in a culture
system comprising the one or more member of TGF.beta. superfamily
of growth factors. The second culture system may also comprise NA,
i.e. may be the same as the initial culture system, into which the
member of the TGF.beta. superfamily is added. As a result, directed
differentiation into RPE cells is induced.
[0111] In accordance with this embodiment, the hSCs in the initial
cell culture are cultured in the NA comprising culture system for
at least the time period required for hSCs differentiation to
initiate. In accordance with one particular embodiment, the cell
culture system is cultivated in the NA comprising culture system
for several days, preferably at least two days and preferably for
at least one week, more preferably, at least two weeks.
[0112] Without being bound by theory, it is stipulated by the
inventors that NA induces the directed differentiation process
which is also accelerated in its progression as compared to
spontaneous differentiation (namely, that occurring in the absence
of NA exposure or exposure to NA in combination with TGF.beta.
member). It has been shown herein that in the directed
differentiation, undifferentiated stem cells are more rapidly
eliminated from the culture system. Hence, NA is used in a culture
system of differentiating hSCs as a mean to promote and accelerate
the directed differentiation process, and for the complete
elimination of undifferentiated stem cells by thus preventing
potential complications such as teratoma tumor formation from the
presence of undifferentiated cells after transplantation.
[0113] It has been shown herein that exposure of the hSCs to NA
followed by exposure to one or more member of the TGF.beta.
superfamily induces differentiation into cells with a different
phenotype as compared to spontaneously differentiating cells (i.e.
in the absence of these factors).
[0114] Further, without being bound by theory, it is assumed by the
inventors that NA induces differentiation into cells that express
receptors to one or more member of the TGF.beta. superfamily (which
are not expressed by spontaneously differentiating stem cells),
thereby allowing directed differentiation into mature and
functional RPE cells. Such receptor expression allows the inductive
effect of the TGF.beta. superfamily member on directed
differentiation of the differentiating cells in the culture towards
RPE fate, namely, towards mature and functional RPE cells.
[0115] As indicated above, there is a variety of members of the
TGF.beta. superfamily of growth factors. For example, the growth
factor in accordance with the invention may be one or more of the
following TGF.beta.1, TGF.beta.2, TGF.beta.3, activin A, activin B,
activin AB, nodal, anti-mullerian hormone (AMH), BMP3, BMP4, BMP5,
BMP6, BMP7 or growth and differentiation factor (GDF). However,
preferably, the growth factor of the TGF.beta. superfamily is
TGF.beta.3 or TGF.beta.1 or activin A or the combination of
same.
[0116] The basic medium in accordance with the invention is any
known cell culture medium known in the art for supporting cells
growth in vitro, typically, a medium comprising a defined base
solution, which includes salts, sugars, amino acids and any other
nutrients required for the maintenance of the cells in the culture
in a viable state. Non-limiting examples of commercially available
basic media that may be utilized in accordance with the invention
comprise Neurobasal.TM., KO-DMEM, DMEM, DMEM/F12, Cellgro.TM. Stem
Cell Growth Medium, or X-Vivo.TM.. The basic medium may be
supplemented with a variety of agents as known in the art dealing
with cell cultures. The following is a non-limiting reference to
various supplements that may be included in the culture system to
be used in accordance with the present disclosure: [0117] serum or
with a serum replacement containing medium, such as, without being
limited thereto, knock out serum replacement (KOSR), Nutridoma-CS,
TCH.TM., N2, N2 derivative, or B27 or a combination; [0118] an
extracellular matrix (ECM) component, such as, without being
limited thereto, fibronectin, laminin and gelatin. The ECM may them
be used to carry the one or more members of the TGF.beta.
superfamily of growth factors; [0119] an antibacterial agent, such
as, without being limited thereto, penicillin and streptomycin;
[0120] non-essential amino acids (NEAA), [0121] neuortrophins which
are known to play a role in promoting the survival of SCs in
culture, such as, without being limited thereto, BDNF, NT3,
NT4.
[0122] Once the cells are promoted into the RPE fate, the RPE cells
may be withdrawn/harvested from the culture by known methods, for
use in various applications.
[0123] The present disclosure also provides RPE cells obtained by
directed differentiation of hSC in the presence of one or more
members of the TGF.beta. superfamily. In accordance with one
embodiment, the RPE cells are obtained by the method of the
invention.
[0124] Further to the above, the RPE cells produced by the directed
differentiation according to the present disclosure have specific
properties in comparison to RPE cells that develop during
spontaneous differentiation. [0125] The differentiating cells have
the potential to respond to TGF.beta. signaling in their
development and differentiation. [0126] The resulting RPE cells are
mature cells (terminally differentiated); [0127] The mature RPE
cells display darker pigmentation in comparison to RPE cells formed
during spontaneous differentiation. [0128] The mature RPE cells
express significantly higher levels of transcripts of markers of
mature RPE cells such as bestrophin, and RPE65 as compared to their
expression in RPE cells produced by spontaneous differentiation. In
this connection, reference is made, for example, to FIGS. 9J, 11M
and 11K showing the expression of bestrophin in spontaneous
differentiation (no NA) as compared to differentiation in the
presence of NA (FIG. 9J) and the augmenting effect of activin A on
directed differentiation (FIGS. 11K, 11M and 4E). Further reference
is made to FIGS. 1B and 9I showing the expression of RPE65 in
spontaneous differentiation (no NA) as compared to differentiation
in the presence of NA and further as compared to the augmenting
effect of activin A in FIG. 4D. [0129] In electron microscope (EM)
analysis the RPE cells display morphological characteristics of
mature authentic RPE cells that are not demonstrated within
RPE-like cells that were derived from spontaneously differentiating
hSC such as apical villi, tight junctions, and basal membrane.
[0130] The RPE cells produced by the method of the present
disclosure may be used for large scale and/or long term cultivation
of such cells. To this end, the method of the invention is to be
performed in bioreactors suitable for large scale production of
cells, and in which undifferentiated hSCs are to be cultivated in
accordance with the invention. General requirements for cultivation
of cells in bioreactors are well known to those versed in the
art.
[0131] Alternatively, the RPE cells produced by the method of the
present disclosure may be expanded after their derivation. For
expansion, they are dissociated, plated at low density on an extra
cellular matrix, preferably poly-D-lysine and laminin, and cultured
in serum-free KOM with NA. Under these culture conditions, the
pigmented cells loose pigmentation and acquired a fibroid-like
morphology. Following further prolonged culture and proliferation
into high-density cultures, the cells re-acquired the
characteristic polygonal shape morphology and pigmentation of RPE
cells.
[0132] The RPE cells may be expanded in suspension or in a
monolayer. The expansion of the RPE cells in monolayer cultures may
be modified to large scale expansion in bioreactors by methods well
known to those versed in the art.
[0133] It would be well appreciated by those versed in the art that
the derivation of RPE cells from hSC is of great benefit. They may
be used as an in vitro model for the development of new drugs to
promote their survival, regeneration and function. hSC-derived RPE
cells may serve for high throughput screening for compounds that
have a toxic or regenerative effect on 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.
[0134] The RPE cells may also serve as an unlimited source of RPE
cells for transplantation, replenishment and support of
malfunctioning or degenerated RPE cells in retinal degenerations.
Furthermore, genetically modified RPE cells may serve as a vector
to carry and express genes in the eye and retina after
transplantation.
[0135] Thus, in accordance with a further aspect of the present
disclosure there is provided a method of transplanting RPE cells
into a subject's eye, the method comprises:
[0136] (a) providing a cell culture comprising hSCs;
[0137] (b) culturing cells in a culture system comprising a basic
medium supplemented with one or more member of TGF.beta.
superfamily whereby the hSCs are promoted to differentiate into RPE
cells;
[0138] (c) harvesting RPE cells from said cell culture; and
[0139] (c) transplanting said differentiated RPE cells into said
subject's eye.
[0140] Harvesting of the cells may be performed by various methods
known in the art. Non-limiting examples include mechanical
dissection and dissociation with papain. Other methods known in the
art are also applicable.
[0141] The hSCs-derived RPE cells may be transplanted to various
target sites within a subject's eye. In accordance with one
embodiment, the transplantation of the RPE cells is to the
subretinal space of the eye, which is the normal anatomical
location of the RPE (between the photoreceptor outer segments and
the choroids). In addition, dependant upon migratory ability and/or
positive paracrine effects of the cells, transplantation into
additional ocular compartments can be considered including the
vitreal space, the inner or outer retina, the retinal periphery and
within the choroids.
[0142] Further, transplantation may be performed by various
techniques known in the art. Methods for performing RPE transplants
are described in, for example, U.S. Pat. Nos. 5,962,027, 6,045,791,
and 5,941,250 and in Eye Graefes Arch Clin Exp Opthalmol March
1997; 235(3):149-58; Biochem Biophys Res Commun Feb. 24, 2000;
268(3): 842-6; Opthalmic Surg February 1991; 22(2): 102-8. Methods
for performing corneal transplants are described in, for example,
U.S. Pat. No. 5,755,785, and in Eye 1995; 9 (Pt 6 Su):6-12; Curr
Opin Opthalmol August 1992; 3 (4): 473-81; Ophthalmic Surg Lasers
April 1998; 29 (4): 305-8; Ophthalmology April 2000; 107 (4):
719-24; and Jpn J Ophthalmol November-December 1999; 43(6): 502-8.
If mainly paracrine effects are to be utilized, cells may also be
delivered and maintained in the eye encapsulated within a
semi-permeable container, which will also decrease exposure of the
cells to the host immune system (Neurotech USA CNTF delivery
system; PNAS Mar. 7, 2006 vol. 103(10) 3896-3901).
[0143] In accordance with one embodiment, transplantation is
performed via pars pana vitrectomy surgery followed by delivery of
the cells through a small retinal opening into the sub-retinal
space or by direct injection. Alternatively, cells may be delivered
into the subretinal space via a trans-scleral, trans-choroidal
approach. In addition, direct trans-scleral injection into the
vitreal space or delivery to the anterior retinal periphery in
proximity to the ciliary body can be performed.
[0144] The RPE cells may be transplanted in various forms. For
example, the RPE cells may be introduced into the target site in
the form of cell suspension, or adhered onto a matrix,
extracellular matrix or substrate such as a biodegradable polymer
or a combination. The RPE cells may also be transplanted together
(co-transplantation) with other retinal cells, such as with
photoreceptors.
[0145] Thus, the invention also pertains to a composition
comprising hSCs-derived RPE cells obtained by the method of the
invention. The composition is preferably such suitable for
transplantation into the eye.
[0146] Various eye conditions may be treated or prevented by the
introduction of the RPE cells obtained by the method of the
invention to a subject's eye. The eye conditions may include
retinal diseases or disorders generally associated with retinal
dysfunction, retinal injury, and/or loss of retinal pigment
epithelium. A non-limiting list of conditions which may be treated
in accordance with the invention comprises retinitis pigmentosa,
lebers congenital amaurosis, hereditary or acquired macular
degeneration, age related macular degeneration (AMD), Best disease,
retinal detachment, gyrate atrophy, choroideremia, pattern
dystrophy as well as other dystrophies of the RPE, Stargardt
disease, RPE and retinal damage due to damage caused by any one of
photic, laser, inflammatory, infectious, radiation, neovascular or
traumatic injury.
[0147] Without being bound by theory, the transplanted RPE cells
may exert their therapeutic effect through multiple mechanisms. One
mechanism is trophic supportive effect promoting the survival of
degenerating photoreceptors or other cells within the retina. RPE
cells derived from hSCs by the methods of this disclosure and in
the presence of a member of the TGF.beta. super family are capable
of preserving the photoreceptors adjacent to them potentially by a
trophic effect.
[0148] The transplanted RPE cells may also exert their effect
through a regeneration mechanism replenishing mal functioning
and/or degenerating host RPE cells. RPE cells derived from hSCs by
the methods of this disclosure and in the presence of a member of
the TGF.beta. super family can replenish mal functioning host RPE.
The transplanted cells are mature and have the functional
capability of phagocytosis of shed outer segments of photoreceptors
which include rhodopsin.
[0149] As mentioned above the RPE cells derived from hSCs by the
methods of this disclosure and in the presence of a member of the
TGF.beta. super family are mature and as such they do not
proliferate in vivo after transplantation. Therefore, RPE cells
derived from hSCs by the methods of this disclosure are safer for
transplantation therapy and carry a reduced risk for development
into teratoma tumors or tumors of proliferating precursor
cells.
[0150] As used herein, the term "treating" or "treatment" refers to
the therapeutic as well as the prophylactic effect of the
hSC-derived RPE cells of the invention on a subject's eye
condition, the effect may generally include, amelioration of
symptoms associated with the conditions, lessening of the severity
or curing the condition, more specifically, the effect may include
reversal of damage caused to the treated subject's retina and RPE,
improved function of the subject's retina, rebuilding of the
subject's retina and RPE by replacement and/or support of failing
host retinal and RPE cells, directly or by paracrine effect as well
as to the prophylactic effect which may be exhibited by the
attenuation, inhibition or cessation in damage caused to the
subject's retina as a result of the condition.
[0151] As used in the specification and claims, the forms "a", "an"
and "the" include singular as well as plural references unless the
context clearly dictates otherwise. For example, the term "a growth
factor" includes one or more growth factors, and the term "growth
factors" includes one growth factor as well as more than one growth
factor.
[0152] As used herein, the term "or" means one or a combination of
two or more of the listed choices. Furthermore, the use of the
phrase "selected from . . . " followed by a list of choices
separated by the term "and" includes one or a combination of two or
more of the listed choices.
[0153] Further, as used herein, the term "comprising" is intended
to mean that the methods or composition includes the recited
elements, but does not exclude 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 will 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 amounts of other elements. Embodiments defined by each of
these transition terms are within the scope of this invention.
[0154] 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%, 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.
SOME EXEMPLARY EMBODIMENTS
Materials and methods
HES Cell Culture
[0155] Human ESC (HES1 cell line) and hESCs engineered by
lentiviral vector to constitutively express eGFP [Gropp M, Itsykson
P, Singer O, Ben-Hur T, Reinhartz E, Galun E, and Reubinoff B E.
Stable genetic modification of human embryonic stem cells by
lentiviral vectors. Molecular Therapy 7:281-7 (2003)] were cultured
on human foreskin fibroblasts feeder layers in KO medium (KOM)
consisting of 86% KO-DMEM (Gibco, Invitrogen, Gaithersburg, Md.),
14% KOSR (Gibco), 1 mM glutamine, 1% nonessential amino acids, 50
units/ml penicillin (Gibco), 50 .mu.g/ml streptomycin, (Gibco) and
4 ng/ml bFGF (R&D Systems, Inc., Minneapolis, Minn.). hES cells
were weekly passaged with type IV collagenase (1 mg/ml; Gibco) and
plated onto a fresh feeder layer. A week before induction of
differentiation, the cells were passaged by dissociation into
nearly a single-cell suspension with Ca/Mg.sup.++-free PBS
supplemented with 0.05% EDTA (Biological Industries, Beit Haemek,
Israel) and were re-plated on the feeders.
EB Formation in Suspension Culture
[0156] Six-eight days after plating the hES cells that were
dissociated into single cells as above; they were removed from the
feeders by treatment with type IV collagenase. The clumps were
cultured for various periods up to 12 weeks in suspension within
bacteriological dishes precoated with 0.1% low melting temperature
agarose in KO medium (KOM) consisting of 86% KO-DMEM, 14% KOSR, 1
mM glutamine, 1% nonessential amino acids, 50 units/ml penicillin,
and 50 .mu.g/ml streptomycin, in the presence or absence of 10 mM
nicotinamide (NA) (Sigma, St. Louis, Mo., USA). In some experiments
the medium used was Neurobasal.TM. medium (Gibco) supplemented with
N2 supplement (1:100) (Gibco) (NN medium), which was substituted
after 1 week with DMEM/F12 (Gibco) supplemented with B27 (1:50)
(Gibco).
Differentiation of hESCs into RPE Cells in the Presence of
TGF-.beta. Growth Factors or Inhibitors
[0157] Human ESCs were allowed to differentiate as free-floating
clusters in KOM as above for up to six weeks in the presence of
nicotinamide (NA) 10 mM. After the first week or 2 weeks of
differentiation, cultures were supplemented with activin A 20-180
ng/ml (PeproTech Inc, Rocky Hill, N.J.), with TGF.beta.3 (1 ng/ml;
R&D Systems Inc, Minneapolis, Minn.) with TGF.beta.1 (1
ng/ml-20 ng/ml; R&D Systems Inc) or with SB431542 (5 .mu.M-50
.mu.M, Sigma). Control cultures were supplemented with NA
alone.
[0158] Human ESCs in suspension in KOM were also supplemented after
one week with the bone morphogenetic protein (BMP) antagonist
noggin (700 ng/ml R&D Systems Inc, Minneapolis, Minn.) in the
presence and absence of NA, or during the 3.sup.rd and 4.sup.th
weeks with TGF.beta. (20 ng/ml PeproTech Inc) in the presence of NA
and allowed to differentiate up to an age of 6 weeks as
free-floating clusters in suspension.
Description of Expansion of the RPE Cells
[0159] To expand the RPE cells, the pigmented clusters were gently
mechanically dissociated into small clamps and plated at low
density on poly-D-lysine ((30-70 kDa, 10 .mu.g/ml) and laminin (4
.mu.g/ml), and cultured in KOM with NA. Under these culture
conditions, the pigmented cells lost pigmentation and acquired a
fibroid-like morphology. Following further culture for 1.5 month
and proliferation into high-density cultures, the cells re-acquired
the characteristic polygonal shape morphology and pigmentation of
RPE cells.
[0160] Immunostaining and real-time RT-PCR were performed on all
cultures as described below.
Indirect Immunofluorescent Staining of Differentiated Cells within
Clusters
[0161] To characterize the immunophenotype of cells within the
aggregates, the clusters cultivated for 2, 4, 6 or 8 weeks were
gently dissociated either with 0.04% trypsin/0.04% EDTA or with
Papain Dissociation System (Worthington Biochemical, Lakewood,
N.J.), and the resulting small clumps and single cells were plated
in KO medium supplemented with NA on poly-D-lysine (30-70 kDa,
10-20 .mu.g/ml) alone or supplemented with either laminin (4
.mu.g/ml) or fibronectin (10-20 .mu.g/ml; all from Sigma, St.
Louis, http://www.sigmaaldrich.com). The cells were fixed with 4%
paraformaldehyde after 2 hours and examined for the expression of
nestin (1:200), polysialic acid NCAM (PSA-NCAM) (1:100), Musashi
(1:200; all from Chemicon, Temecula, from CA), Pax6 (DSHB, 1:100 or
Chemicon, 1:250), Otx2 (Chemicon, 1:200), MiTF (Lab Vision
Corporation, Fremont, Calif.; mouse IgG.sub.1, 1:50).
[0162] For immunostaining of enriched preparations of pigmented
cells, the pigmented (Brown) clusters of cells within the floating
clumps, that differentiated 8-10 weeks, were mechanically dissected
and isolated by glass micropipettes or scalpel blades (No 15;
Swann-Morton Sheffield, Eng).
[0163] The isolated clusters that were enriched for pigmented cells
were further dissociated into smaller clumps mechanically by
trituration with/without the aid of trypsin (0.025%, 3 mM EDTA in
PBS) digestion or papain dissociation (Papain Dissociation System;
Worthington Biochemical Corporation, Lakewood, N.J.). The small
clusters of cells were plated on poly-D-lysine-coated (30-70 kDa,
10 .mu.g/ml; Sigma) and laminin-coated (4 .mu.g/ml; Sigma) glass
coverslips and cultured for an additional 3-5 weeks in the culture
medium used for suspension culture of the hESC clusters.
Differentiated cells within the outgrowth were fixed with 4%
paraformaldehyde for 30 minutes at room temperature. For
immunostaining with anti-intracellular marker antibodies, cell
membranes were permeabilized with 0.2% Triton X100 (Sigma) in PBS
for 30 minutes, supplemented with normal goat serum (5%, Biological
Industries) for blocking. The cells were incubated with the
following primary antibodies: 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; mouse IgG.sub.1, 1:150);
anti-ZO-1 (Zymed Laboratories Inc., San Francisco, Calif.; rabbit
polyclonal, 1:10); anti-Ki67 (Dako Denemark A/S; 1:50) and
anti-CRALBP (kindly provided by John C. Saari, University of
Washington, Seattle; rabbit polyclonal, 1:100). The cells were also
incubated with Phalloidine (1:200 Sigma).
[0164] Primary antibody localization was performed by using
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse
immunoglobulins (Dako Denmark A/S; 1:20-1:50), goat anti-mouse IgG
conjugated to Cy.sup.TH3 (1:500) (Jackson ImmunoResearch
Laboratories Inc, West Grove, Pa.), rabbit anti-goat igG conjugated
to Cy2 (1:200; Jackson ImmunoResearch Laboratories Inc) and swine
anti-rabbit Ig conjugated to fluorescein isothiocyanate (FITC)
(Dako; 1:50).
Analysis of hESC Clusters by RT-PCR and Real-Time PCR
[0165] Total RNA was extracted from hESCs grown under serum-free
conditions (1 week after passage), and at sequential time points up
to 8 weeks during culturing of hESC-derived clusters in the
presence or absence of 10 mM nicotinamide and with or without
supplementation with TGF.beta.-superfamily growth factors or
antagonists. The RNA was isolated using TRIzol reagent (Invitrogen,
http://www.invitrogen.com) or TRI-Reagent (Sigma). cDNA synthesis
was carried out using Moloney murine leukemia virus reverse
transcriptase (M-MLV RT) and random primers, according to the
manufacturer's instructions (Promega Corporation, Madison, Wis.,
http://www. promega.com). Polymerase chain reaction (PCR) was
carried out using standard protocols with Taq DNA Polymerase
(Gibco-BRL). Amplification conditions were as follows: denaturation
at 94.degree. C. for 15 seconds, annealing at 55.degree. C. for 30
seconds, and extension at 72.degree. C. for 45 seconds. The number
of cycles varied between 18 and 40, depending on particular mRNA
abundance. Primer human sequences for identifying human gene
transcripts (forward and reverse 5'-3') and the length of the
amplified products were as follows (SEQ ID NOs. 1-12):
TABLE-US-00001 MiTF-A (GAGCCATGCAGTCCGAAT, GACATGGCAAGCTCAGGACT;
486 bp); RPE65 (GCTGCTGGAAAGGATTTGAG, CAGGCTCCAGCCAGATAGTC; 231
bp); Bestrophin (GAATTTGCAGGTGTCCCTGT, ATCCTCCTCGTCCTCCTGAT; 214
bp); CRALBP (AGCTGCTGGAGAATGAGGAA, CAAGAAGGGCTTGACCACAT; 218 bp)
MERTK (AAGTGATGTGTGGGCATTTG, TCTAAGGGATCGGTTCTCCA, 189 bp); ACTRIA
(AATGTTGCCGTGAAGATCTTC, CTGAGAACCATCTGTTGGGTA; 699 bp); ACTRIB
(CACGTGTGAGACAGATGGG, GGCGGTTGTGATAGACACG; 346 bp); ACTRIIA
(AACCATGGCTAGAGGATTGGC, CTTTCACCTACACATCCAGCTG; 551 bp); ACTRIIB
(CACCATCGAGCTCGTGAAG, GAGCCCTTGTCATGGAAGG; 611 bp); Activin A
(CTTGAAGAAGAGACCCGAT; CTTCTGCACGCTCCACCAC; 262 bp); .beta.-actin
(TTCACCACCACGGCCGAGC, TCTCCTTCTGCATCCTGTCG; 351 bp); GAPDH
(AGCCACATCGCTCAGACACC; GTACTCAGCGCCAGCATCG; 301 bp).
[0166] For Real-time PCR the levels of transcripts were monitored
using TaqMan primers and probes that were derived from the
commercially available TaqMan.RTM. Assays-on-Demand Gene Expression
Products (Applied Biosystems, Foster City, Calif.): Oct4, ID
Hs01895061; Musashi, ID Hs01045894; Pax6, ID Hs00240871; Six3, ID
Hs00193667; Rx1, ID Hs00429459; Chx10, ID Hs01584048; MiTF-A, ID
Hs01115553; MiTF-total, ID Hs01115557; Bestrophin, ID Hs00188249;
RPE65, ID Hs00165642; Sox10, ID Hs00366918; Crx, ID Hs00230899.
Quantitative PCR analysis was performed using an ABI Prism 7000HT
and ABI Prism 7900HT Sequence Detection Systems and TaqMan.RTM.
Universal PCR Master Mix (Applied Biosystems) according to the
manufacturer's protocol. Housekeeping gene .beta.-glucuronidase
(GusB, assay ID Hs99999908) was selected as an internal reference
for normalization in the Real-Time RT-PCR quantification analysis
and the relative expression level of each gene is shown as a
relative value when the expression level of day 0 (or untreated
cells) was set at 1. Amplification reactions were carried out in
duplicates or triplicates according to manufacturer's protocol
(Applied Biosystems).
Transmission Electron Microscopy and Phagocytosis of Latex
Beads
[0167] Human ESC-derived clusters were cultured in suspension in
KOM. The pigmented areas were then separated mechanically and were
processed for transmission electron microscopy. The cells were
fixed with 2% glutaraldehyde and 4% formaldehyde in 0.1M cacodylate
buffer, pH 7.4. After three washes in 0.1M cacodylate buffer the
tissue was post-fixed with 1% osmium tetroxide and 1.5% potassium
fericyanide, dehydrated with increasing concentrations of ethanol
and embedded in Agar 100 resin. Ultra-thin sections cut by an LKB
ultrotome 3 were stained with uranyl acetate and lead citrate.
Micrographs were taken with Tecnai 12 electron microscope
(Phillips, Eindhoven the Netherlands http://www.philips.com)
equipped with a Megaview II CCD camera and an Analysis version 3.0
software (Soft Imaging System, http://www.soft-imaging.net).
[0168] To examine phagocytotic ability, the pigmented clusters were
incubated with 1-.mu.m latex beads (Polysciences Inc., Warrington,
Pa.) at a concentration of 1.0.times.10.sup.9 beads/mL for 18 hours
at 37.degree. C. The pigmented clusters were then washed with PBS+,
dissociated into single cells or small clumps using Papain
Dissotiation System and plated on poly-D-lysine. Following
fixation, the membranes of the cells were stained with red
fluorescent die, PKH (Sigma). The phagocytosis was analyzed using
confocal microscope (Olympus Fluoview FV1000).
Flow Cytometry
[0169] Flow cytometric analysis was performed to determine the
number of PSA-NCAM and TRA-1-60 positive cells at different time
points within the hESC-derived clusters differentiating with or
without nicotinamide supplementation. The clusters were dissociated
with 0.04% Trypsin/0.04% EDTA. The single cells were then stained
with anti-PSA-NCAM or anti-Tra-1-60 antibodies (both from Chemicon;
1:100), detected with goat anti-mouse immunoglobulins conjugated to
FITC (Dako; 1:100), and counterstained with the cell viability dye
Propidium iodide (0.005 mg/ml; Sigma) Control cells were incubated
only with a secondary antibody. The cell-associated
immunoreactivity was analyzed with FACScalibur (Becton Dickinson
Immunocytometry Systems), using CellQuest software.
Intravitreal and Subretinal Transplantation of hESC-Derived
Differentiated RPE Cells
[0170] For intra-ocular transplantation, hESCs engineered to
express eGFP [as previously described in Gropp et al., Stable
genetic modification of human embryonic stem cells by lentiviral
vectors. Molecular Therapy 2003; 7: 281-7) were used to generate
RPE cells in culture as described above. Briefly, clusters enriched
with pigmented cells were mechanically isolated by dissection
following differentiation for 6-8 weeks in the presence of NA alone
or in the presence of NA supplemented with activin A. To allow
injection through a small bore glass capillary, the clumps were
further 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.
[0171] Fifteen adult albino rats (body weight 230-250 g) and over
100 1-3 weeks outbred dystrophic RCS rats were used for intraocular
transplantation. In RCS rats, a mutation in the Mertk gene causes
RPE dysfunction which leads to retinal degeneration over the first
few months of life. 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 of the Hebrew University-Hadassah Medical
School.
[0172] For transplantation (as well as for eletroretinographic
recordings) 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), approximately
100,000 cells in 4 .mu.L of medium were injected into the vitreous
or into the subretinal space via a trans-scleral, transchoroidal
approach through a glass capillary coupled to a pneumatic
Pico-injector (PLI-100; Medical System Corp., Greenvale, N.Y.,
http://www.medicalsystems.com). Fellow, non-injected eyes served as
one type of control. As an additional control, eyes were injected
with saline (Sodium Chloride Injection BP, 0.9%, B. Braun Melsungen
AG, Melsungen, Germany).
[0173] 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, Basel, Switzerland) in their drinking water at
a concentration of 210 mg/l.
In-Vivo and Ex-Vivo Imaging of Transplanted Cells
[0174] To monitor survival and location of the transplanted cells
in-vivo, anesthetized animals were imaged using a color fundus
camera (Zeiss, Germany) and fluorescence of the GFP-expressing
cells was detected using fluorescein filters on a scanning laser
ophthalmoscope (Heidelberg HRA, Germany). In some eyes, location of
the GFP-positive grafts was also determined ex-vivo in eye-cup
preparations using a fluorescent microscope (Canon, Japan).
Assessment of Host Retinal Function Following Intra-Ocular
Transplantation of hESC-Derived RPE Cells
[0175] Four to six weeks after transplantation, retinal function
was assessed in transplanted and control RCS rat eyes by
electroretinography (ERG). Full field ERGs were recorded following
overnight dark adaptation. Animals were anesthetized with Ketamine
and Xylazine in dim red light, and pupils were dilated with
tropicamide and phenyephrine. Monopolar rat ERG lens electrodes
(Medical Workshop, Amsterdam, Netherlands) were placed on each eye
following additional topical anesthesia, with a reference electrode
and a ground electrode placed on the tongue and tail respectively.
A commercial computerized ERG system (LKC technologies, UTAS 3000)
was used to record retinal responses to full field stimuli
generated using a Xenon photostrobe flash (Grass, PS-22) mounted on
a Ganzfeld bowl. Dim blue flashes under scotopic conditions were
used to elicit a largely rod-driven response. At stronger stimulus
intensities of blue, and with white flashes in the dark-adapted
state, mixed cone-rod responses were recorded. Under light-adapted
(photopic) conditions, with white flashes on a rod-suppressing 34
cd/m.sup.2 white background, 1 Hz and 16 Hz cone responses were
generated. Signals were filtered between 0.3-500 Hz, and signal
averaging was used.
Histological and Immunohistochemical Evaluation of Transplanted
Eyes
[0176] Animals were sacrificed 4-8 weeks after transplantation and
eyes enucleated for histological and immunohistochemical
examination. Following fixation in Davidson solution, 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 and quantification. For indirect
immunofluorescent studies, to characterize state of differentiation
of the transplanted cells, specimens were de-paraffinized 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 (BSA), 0.1% Triton X100
(Sigma-Aldrich), and 3% normal goat or normal donkey serum.
Subsequently, sections were incubated for 1 hour in a humidified
chamber with appropriate combinations of the following primary
antibodies: anti-green fluorescent protein (anti-GFP), conjugated
with fluorescein (FITC) or rhodamine (TRITC) (Santa Cruz
Biotechnology, Inc, Santa Cruz, Calif.; mouse monoclonal, 1:100);
anti-RPE65 (Novus Biologicals, Littleton, Colo.; mouse IgG.sub.1,
1:100); anti-Bestrophin (Novus Biologicals; mouse IgG.sub.1,
1:100); anti-ZO-1 (Zymed Laboratories Inc., San Francisco, Calif.;
rabbit polyclonal, 1:100); and anti-rhodopsin (Santa Cruz
Biotechnology, Inc, Santa Cruz, Calif.; rabbit polyclonal, 1:100).
Primary antibody localization was performed after washing in PBS by
using Cy.TM. 2-conjugated goat anti-rabbit IgG (1:200), Cy.TM.
2-conjugated goat anti-mouse IgG (1:200), Cy.TM. 3-conjugatedgoat
anti-rabbit IgG (1:200), Cy.TM. 2-conjugated donkey anti-mouse IgG
(1:200), Cy.TM. 5-conjugated donkey anti-rabbit IgG (1:200; all
from Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa.).
Nuclei were counterstained with 4,6-diamidino-2-phenylindole
(DAPI)--containing mounting medium (Vector Laboratories,
Burlingame, Calif.) or with propidium iodide 1 .mu.g/ml (BioLegend,
San Diego, Calif.). To determine the specificity of the
antigen-antibody reaction, corresponding negative controls with an
irrelevant isotype-matched antibody were performed. An Olympus BX41
microscope equipped with a DP70 digital camera (Olympus, Japan) was
used for fluorescent and light microscopy imaging. Confocal images
were collected on an Olympus Fluoview 300 (FV300) confocal
microscope (Olympus, Japan) built around an IX70 inverted
microscope. 488-nm Ar, 543 HeNe-Green, and 633 HeNe Red lasers were
used in combination with Nomarski optics.
Quantification of Photoreceptor Layer Rescue in Vicinity to RPE
Grafts
[0177] To quantify the effect of hESC-derived RPE transplantation
on the degenerating host retina, high resolution microscopic images
of hematoxylin and eosin stained sections were obtained and
montages of the full length of the retina constructed using
Photoshop software (Adobe, USA). Total retinal thickness, thickness
of the outer nuclear (photoreceptor) layer as well as thickness of
the inner- and outer-segments layer were measured in proximity to
subretinal grafts of hESC-derived RPE cells using the J-image
program (NIH). These were compared to measurements obtained in the
corresponding opposite side of the retina, distant from the graft.
Since the degenerative process in RCS rats is location-dependant,
thickness was measured in areas that were of equal distance from
the cilliary body. In each area, at least three equally spaced
measurements were averaged.
Results
Characterization of Differentiated RPEs
[0178] Differentiation of hESCs was induced by culturing them as
free-floating clusters in KO medium supplemented with NA. Under
these culture conditions defined areas highly enriched for
pigmented cells developed within the differentiating clusters, as
shown in FIG. 3A. These pigmented areas appeared after 4 weeks of
differentiation and after 8 weeks 72.9.+-.2.5% of the clusters had
pigmented areas. Pigmented areas were not observed after 4 weeks of
differentiation in the absence of NA supplementation, and in these
conditions only 13.1.+-.4.8% developed pigmented areas after 8
weeks (FIGS. 8A, and 8B). Thus, NA treatment augmented/promoted
differentiation within hESC clusters into pigmented cells as
compared to spontaneously differentiating hESC clusters.
[0179] Within clusters that differentiated 8 weeks in the presence
of NIC, 5.7.+-.1.0% of the cells were pigmented, 5.4.+-.1.1%
expressed the early RPE marker MiTF and in most cases the
expression of MiTF correlated with pigmentation (FIG. 8C).
Partially dissociated and plated clusters of differentiated hESCs
developed, among other types of differentiated cells, into colonies
composed of monolayers of pigmented cells, as shown by a dark field
micrograph (FIG. 3D) and a phase contrast image (FIG. 3E). The
cells within these colonies assumed a polygonal shape and formed
"cobble stone"-like sheets of cells with tight junctions between
them (FIG. 3E), features that are highly characteristic of native
RPE cells. The F-actin distribution within the cells was adjacent
to their membranes, like authentic RPE cells as demonstrated by
staining with Phalloidin (FIG. 10A). The pigmented, RPE cells
co-expressed the RPE markers Otx2 (FIG. 3B) and MiTF-A (FIG. 3B and
FIG. 3F), as well as ZO-1 (FIG. 3G), Bestrophin (FIG. 3H), RPE65
(FIG. 3I), and CRALBP (FIG. 3J).
[0180] After dissociation, plating at low density and cultivation,
the pigmented cells lost pigmentation and acquired a fibroid-like
morphology, as shown by phase contrast imaging (FIGS. 3K, and 10B).
Following further prolonged cultivation and proliferation into
high-density cultures, the cells re-acquired the polygonal shape
morphology and pigmentation of RPE cells (FIGS. 3L, and 10C).
[0181] Electron microscopy (EM) analysis demonstrated that the
hESC-derived pigmented cells had morphologic characteristics of
native RPE cells including microvilli on their apical side (FIG.
10D), and basal membrane on their basal side (FIG. 10E). The cells
contained melanin granules (FIG. 10D) and were attached by tight
junctions (FIG. 10F).
[0182] One of the most important functions of RPE cells is
phagocytosis of photoreceptors shed outer segments. To examine
whether the hESC-derived pigmented cells had phagocytic capability,
they were incubated with 1-.mu.m fluorescent latex beads. The three
confocal fluorescent images demonstrate serial z axis slices (FIGS.
10H-10J). Confocal microscope analysis showed that the putative RPE
cells were capable of phagocytosis of the fluorescent beads (FIGS.
10G-10J).
Differentiation-Inductive Effect of Nicotinamide
[0183] Differentiation of hESCs toward an RPE fate was examined by
culturing them as free-floating clusters in KOM with or without NA
(without providing spontaneously differentiating clusters as
controls). After 6 weeks of differentiation, the level of
expression of markers of RPE cells, MiTF-A and RPE65, was
significantly enhanced in the presence of NA, as determined by
Real-time RT-PCR (FIGS. 1A, and 1B; respectively). The expression
of MiTF-A increased nearly two-fold in the presence of NA, while
the expression of RPE65 increased nearly 30-fold. Since most
pigmented cells co-expressed MITF-A (FIG. 8C), it appeared that in
the presence of NA there was a significant and prominent increase
in the level of expression of RPE65 per pigmented cell. Thus, in
addition to its inductive effect towards RPE fate, NA also promoted
the maturation of the RPE cells and had an effect on the phenotype
of the cells. Q-PCR analysis at sequential time points between 2
and 6 weeks showed an increase in MiTF-A and RPE65 expression
levels (FIGS. 1C and 1D, respectively), in the presence of NA. The
expression of the markers was up-regulated after four weeks of
differentiation, and the levels of expression continued to increase
during the following four weeks (final two weeks not shown).
Similar increased expression of the RPE markers Bestrophin, CRALBP
and Mertk was shown by RT-PCR analysis of cells in plated,
pigmented clusters (FIG. 1E).
[0184] In order to find whether the development of RPE cells in
vitro recapitulates the key developmental steps of RPE in vivo,
time course experiments were performed including analyses of the
expression within the hESC clusters of key markers during RPE
development. Clusters differentiating in the presence or absence of
NA were analyzed by Real-time PCR for the expression of markers of
undifferentiated hESCs, early neural differentiation, retinal and
RPE development (FIGS. 9A-9L). First it was demonstrated that the
expression of Oct4 (FIG. 9A), a marker of undifferentiated hESCs,
declined more rapidly during differentiation in the presence of NA.
In accordance, FACS analysis demonstrated that the expression of
TRA-1-60, a surface membrane marker of undifferentiated cells, also
declined more rapidly in NA treated samples (FIG. 9M). Thus,
differentiation in the presence of NA may be used to eliminate
undifferentiated cells from the culture and may aid in avoiding
teratoma tumor formation after transplantation.
[0185] In addition, NA treatment enhanced the process of early
neural differentiation. In the presence of NA, the expression of
transcripts of the early neural markers Otx2 (FIG. 9B), Pax6 (FIG.
9D) and Musashi (FIG. 9C) was significantly increased after 2, 2-6
and 4-6 weeks of differentiation, respectively. Similar results
were demonstrated at the protein level by FACS analysis of the
expression of PSA-NCAM, a marker of neural precursors (FIG. 9O).
After 4 weeks of differentiation with NA, 81.4.+-.6.3% of the cells
expressed PSA-NCAM as compared to 14.4.+-.5.9 in control clusters.
Indirect immunofluorescence staining confirmed that at 4 weeks, the
majority of cells within the NA-treated clusters acquired a neural
phenotype and expressed PSA-NCAM (74.2.+-.4.1%), nestin
(55.9.+-.10.1%), and Musashi (71.4%; FIG. 9N).
[0186] The expression of transcripts of Rx1 and Six3 (FIGS. 9F and
9E, respectively), which are key regulatory genes of retinal
specification and morphogenesis, was demonstrated after 2 weeks of
differentiation. NA treatment increased the expression of these
genes.
[0187] In the presence of NA the expression of transcripts of the
early RPE marker, MiTF A was induced after 4 weeks of
differentiation (FIG. 9H). The expression of markers of more mature
RPE, Bestrophin and RPE65 was mainly up regulated after 4 and 8
weeks, respectively (FIGS. 9J and 9I, respectively). The expression
of these transcripts was also higher in NA-treated cultures as
compared to spontaneously differentiating hESC clusters as controls
(FIGS. 9I, and 9J). The expression of RPE65 was augmented by more
than a 100 folds further confirming that in addition to inductive
effect NA also had an effect on the phenotype of the cells). To
rule out that the pigmented cells that were obtained are not neural
crest-derived melanocytes, the expression of Sox10 was
demonstrated, which is a developmental marker of these cells, was
low compared to control cells of the M51 melanoma cell line and not
dependent on NA supplementation. Thus the cultures were not
comprised of melanocytes. Thus, it was concluded that NA promotes
the induction of differentiation toward an RPE fate.
[0188] NA treatment also increased the expression of Chx10 which
regulates the proliferation of neural retina retinal progenitors
and of Crx, the marker of photoreceptor progenitors (FIG. 9K).
[0189] In summary, it was concluded by the inventors that the RPE
differentiation process within the hESC clusters went through steps
similar to authentic RPE development in vivo and was augmented by
NA. Furthermore, NA had an effect on the phenotype of the RPE cells
that were obtained. These cells were different from RPE cells that
are obtained after spontaneous differentiation and expressed
markers of mature RPE cells at significant higher levels.
NA Shows an Inductive Effect Regardless of Culture Medium
[0190] NA supplementation increased pigmentation in differentiated
cells cultured in both KO medium and NN/DMEM medium for 12 weeks,
as shown in dark field micrographs of clusters of differentiating
hESCs (FIGS. 2A-2D). In NN medium that was further replaced after a
week by DMEM/F12-B27 (NN/DMEM), as compared to KO medium, in the
presence of NA, the percentage of pigmented hESC clusters from the
total number of clusters was higher, although their size and total
population numbers relative to clusters cultured in KO medium were
smaller. RT-PCR analysis showed that NA supplementation enhanced
the expression of MiTF-A approximately 3-fold in KOM and
approximately 2.5-fold in NN/DMEM medium (FIG. 2E). The expression
of RPE65 was approximately doubled in KOM, and increased nearly
6-fold in NN/DMEM medium, with supplementation of NA versus without
NA (FIG. 2F). Thus, the differentiation-inductive effect of NA is
shown in differentiated RPE cells regardless of the medium in which
hESCs are cultured and differentiation occurs.
The Effect of Members of the TGF.beta. Superfamily on SC
Differentiation
[0191] First analyzed was the expression of activin receptors as
well as activin A in 2 weeks old clusters. Analysis was performed
at this time point since the expression of early eye field markers
is emerging at this time and therefore the differentiating cells
are probably at a developmental stage parallel to early optic
vesicle. It was demonstrated that the expression of the receptors
ACTRIB and ACTRIIB was high in the presence of NA in comparison to
lack or minor expression when the cells were differentiating
without NA (FIG. 11G). Thus NA had an effect on the phenotype of
cells differentiating in its presence.
[0192] Activin A was found to augment differentiation towards RPE
fate. Dark field micrographs of hESC-derived clusters
differentiating for 4 weeks showed the appearance of pigmented
cells at this early stage in the presence of activin (FIG. 11A) It
was further shown that in the presence of NA, activin A
significantly augmented the differentiation of hESCs towards RPE
cells (FIGS. 4A-4B, and 11B-11C). The percentage of clusters that
included pigmented cells (50.7.+-.6.5_vs 17.7.+-.3.2), as well as
the percentage of pigmented cells from the total number of cells
(9.9.+-.1.4 vs 2.4.+-.1.2), was significantly higher when
differentiation was induced in the presence of activin A as
compared to the control cultures supplemented with NA without
activin A (FIGS. 11H and 11I). This result was confirmed with
RT-PCR, which showed that activin A treatment significantly
increased (over five-fold and over four-fold, respectively) the
expression of the markers RPE65 (FIG. 4D) and Bestrophin (FIG. 4E)
which are specific to mature RPE-cells. Furthermore, the
morphological characteristics of clusters of pigmented cells that
developed in the presence of activin A were different. Their
pigmentation was darker and they displayed a very clear demarcation
from surrounding non-pigmented cells (FIG. 4B). The expression of
MiTF-A, a marker which appears earlier during RPE development was
not affected by supplementation with activin A. Thus, activin A,
which is a member of the TGF.beta. superfamily of growth factors,
augments the differentiation of hESCs into RPE cells. The
supplementation of the activin inhibitor, SB431542, significantly
decreased the appearance of pigmented clusters under these
conditions (FIGS. 11E, and 11H). The effect of activin A on RPE
differentiation was studied in various concentrations. The effect
was found to be dose dependent with optimal augmenting effect at
140 ng/ml on the percentage of pigmented cells and the expression
of RPE markers, Bestrophin (FIG. 11K) and RPE65 (FIG. 11L). In most
of the experiments activin A was supplemented for two weeks (weeks
3-4) after the clusters have already differentiated for 2 weeks,
since we found that application at this time period was optimal for
enhancing RPE differentiation. Given the observation that the
expression of markers of early eye development also began after 2
weeks of differentiation (FIGS. 9A-9L), it appeared that activin
augmented the process of eye and RPE development. Moreover, since
the percentage of pigmented cells increased by 5-6 folds in the
presence of activin A, while the expression of markers of mature
RPE cells such as bestrophin increased by about 10 folds, it
appears that activin A had an effect on the maturity and phenotype
of the cells in addition to its inductive effect. This was
supported by the morphological appearance of pigmented cells that
were obtained in the presence of activin A as they were darker and
with sharp demarcation from surrounding cells.
[0193] Q-PCR time-course analysis of the effect of 2 weeks activin
A treatment on gene expression showed that activin A significantly
increased the expression of the retinal progenitor markers Rx1 and
Chx10 as well as the RPE marker Bestrophin. At 4 weeks of
differentiation activin A treatment also increased the expression
level of total MiTF-isoforms (FIGS. 11M-11P).
[0194] The inductive effect of activin A on retinal and RPE
differentiation was observed also with other members of the
TGF.beta. superfamily. Treatment of differentiating clusters with
TGF.beta.3 significantly augmented the expression levels of
transcripts of MiTF-A, which plays a key role in RPE development in
vivo (FIG. 5F). Furthermore, treatment with TGF.beta.1, which is
another member of the TGF.beta. superfamily also significantly
enhanced the appearance of clusters harboring pigmented cells
(FIGS. 11D, and 11H). In contrast, differentiation in the presence
of NA and basic FGF (bFGF) instead of factors from the TGF.beta.
superfamily abolished the appearance of pigmented cells (FIG.
11F).
[0195] In addition, it was shown that bone morphogenetic proteins
(BMPs), which belong to a second sub-family of the TGF.beta.
superfamily, play a role in RPE differentiation of hESCs. As shown
in dark field micrographs, in the presence of the BMP antagonist
noggin, differentiation of hESC clusters into pigmented RPE cells
was blocked (FIG. 5D). Differentiation into pigmented cells was
blocked with supplementation of noggin even when the medium was
also supplemented with NA (FIG. 5C). At the RNA level, Real-Time
RT-PCR demonstrated that noggin significantly reduced the
expression of MiTF-A in both the presence and absence of NA in the
culture medium (FIG. 5E). Thus, BMPs play a role in inducing
differentiation of hESCs to RPE cells.
Differentiated RPE Cells Derived from hESCs May be Used for
Intraocular Transplantation
[0196] Enriched populations of hESC-derived RPE cells engineered to
express eGFP were initially transplanted into the vitreous and
subretinal spaces of albino rats, to facilitate localization of the
pigmented cells. Following intraocular transplantation, the
transplanted pigmented cells could be readily identified in vivo
(FIGS. 6A and 6B). Following enucleation of the eye shown in FIG.
6B, removal of the cornea and lens and isolation of the retina, the
retina showed the main graft as well as additional dispersed
pigmented cells (FIG. 6C). In histologic sections, grafts that
included viable pigmented transplanted cells which also expressed
GFP were present (FIGS. 6D-6G). Transplanted cells could be found
in the vitreal space, in the retina, along the tract of injection,
and also in the subretinal space (FIGS. 6H, 6I, 6O, 6P).
Transplanted RPE cells also migrated from subretinal grafts and
integrated within the RPE layer of host rats (FIG. 6J). In the
grafts, tight junctions, which are characteristic of RPE cells,
were formed, as shown by expression of the tight junction marker
ZO-1 in transplanted eGFP-positive cells (FIG. 6K-6N). Cells within
the grafts also maintained the expression of RPE65, Bestrophin, and
MiTF-A.
hESC-Derived RPE Cells Survive, Integrate and Maintain
Characteristics of Differentiated RPE Following Transplantation
into the Subretinal Space of RCS Rats
[0197] The main body of transplantation experiments were performed
in RCS rats which manifest an RPE and retinal degeneration caused
by a mutation in the mertk gene, in an attempt to examine whether
delivery of hESC-derived RPE cells can modulate the course of
disease.
[0198] The transplanted pigmented cells could be readily identified
in-vivo in the eyes of RCS rats using standard fundus imaging
systems (FIGS. 12A-12C). The hESC-derived, GFP-expressing cells can
be seen to emit fluorescence when fluorescein excitation and
emission filters are used (FIG. 12C). In eye cup preparations
imaged ex-vivo in a fluorescence microscope (FIGS. 12D-12E), large
clusters of subretinal GFP-positive cells can be seen (FIG. 12D) as
well as multiple, dispersed smaller clusters (FIG. 12E).
[0199] Histologic and immunohistochemical evaluation confirmed the
in-vivo and ex-vivo macro observations. The transplanted cells
survived and integrated in the subretinal space, and maintained
expression of proteins that characterize and are often specific to
mature RPE (FIGS. 13 and 14). It is important to note that no
significant inflammation or immune reaction was present, and no
tumors or teratomas were observed in over 100 transplanted eyes.
Sections stained by hemotoxylin and eosin (FIGS. 13A and 13B) show
the subretinal and occasionally intra-retinal location of
transplanted hESC-derived pigmented cells, which appeared in
clusters or as isolated cells (arrows).
[0200] Immunostaining with GFP (FIGS. 13C-13F) confirmed that the
cells are indeed hESC-derived. Grafts were often quite large and
dispersed (FIGS. 13C, and 13E), and pigmented cells co-expressing
GFP were clearly seen (FIGS. 13D, and 13F).
[0201] Immunostaining revealed that large numbers of transplanted
cells within grafts express proteins which characterize mature,
differentiated RPE cells (FIG. 14). This included expression of the
RPE-specific markers RPE65 (FIGS. 14A-14E) and Bestrophin (FIGS.
14F-14J). The cells were also capable of forming tight junctions
(FIGS. 14K-14O), which is an important function of RPE cells and
essential for maintaining the blood-retinal barrier. Left-most
panel in each row shows low-magnification fluorescent image of
grafts co-expressing GFP and the relevant marker. High
magnification confocal images in each row show pigment (by Nomarski
optics) as well as co-expression of GFP and the different markers
at the single-cell level.
hESC-Derived RPE Cells Provide Functional and Structural Retinal
Rescue in Dystrophic RCS Rats
[0202] In the RCS rat model of retinal degeneration, retinal
function is usually severely impaired by 2-3 months of age.
Structurally, corresponding loss and thinning of the retinal outer
nuclear layer (ONL) occurs and it is often reduced to less than 1-2
rows of photoreceptor nuclei at this age. In eyes transplanted with
hESC-derived RPE cells, electroretinographic recordings revealed
significant relative preservation of retinal function as compared
to control untreated or medium-injected eyes (FIG. 7 and FIG.
15).
[0203] Representative ERG responses to a series of white flashes of
increasing intensity in the dark-adapted (DA) state are shown in a
transplanted eye (FIG. 15A) versus its fellow control eye (FIG.
15B). FIG. 15C shows the marked differences in mean amplitudes
between transplanted eyes and the different groups of control eyes.
At the highest intensity, mean DA b-wave amplitude in
RPE-transplanted eyes was 283.3.+-.37.5 (mean.+-.SEM; n=13) versus
158.5.+-.18.1 in fellow non-treated control eyes (n=13, p<0.01)
and 89.9.+-.14.4 in medium-injected eyes (n=5, p<0.01). It is
important to note that there is a trend towards better preservation
of retinal function following transplantation of Activin-A treated
RPE cells (FIG. 15) as compared to the rescue effect achieved
following transplantation of RPE cells derived without activin-A
(FIG. 7).
[0204] Qualitative as well as quantitative assessment of retinal
structure corroborated the functional findings (FIG. 16). Relative
preservation of the photoreceptor (ONL) layer and of the inner and
outer photoreceptor segments (IS+OS) was observed in proximity to
sub-retinal RPE grafts as compared with areas distant from the
grafts (two examples shown in FIG. 16A, 16B). Total retinal
thickness (FIG. 16C) as well as ONL and IS+OS thickness (FIG. 16D)
are significantly increased in vicinity to hESC-derived RPE grafts
(black bars, mean.+-.SEM, n=7) as compared to areas distant from
grafts (gray bars). This type of structural rescue was observed
only in proximity to sub-retinal and deep intra-retinal grafts, and
not when grafts were exclusively intra-vitreal (not shown).
Transplanted hESC-Derived, Activin-A Treated RPE Cells Uptake
Rhodopsin In-Vivo
[0205] One of the key functions of healthy RPE is the uptake and
recycling of shed photoreceptor outer segments, as part of the
renewal process of the photoreceptor. Confocal images of subretinal
transplanted RPE cells show the co-localization of pigment, GFP,
RPE65 and rhodopsin within the same single cells. This suggested
that the transplanted cells have the phenotype of mature RPE and
are able to perform uptake of shed outer segments (which contain
rhodopsin). Note that the native RPE cells of the RCS rat express
RPE65 (FIG. 17C, arrow) but do not express GFP (FIG. 17D, arrow)
and contain minimal amounts of rhodopsin (FIGS. 17B, 17E).
[0206] The above results thus provide evidence that hSCs derived
RPE cells obtained by culturing in a culture system comprising a
member of the TGF.beta. superfamily and preferably in the presence
of NA can be utilized in vivo for transplantation to provide
essentially fully functional RPE cells in the eye.
Sequence CWU 1
1
12138DNAHomo sapiens 1gagccatgca gtccgaatga catggcaagc tcaggact
38240DNAHomo sapiens 2gctgctggaa aggatttgag caggctccag ccagatagtc
40340DNAHomo sapiens 3gaatttgcag gtgtccctgt atcctcctcg tcctcctgat
40440DNAHomo sapiens 4agctgctgga gaatgaggaa caagaagggc ttgaccacat
40540DNAHomo sapiens 5aagtgatgtg tgggcatttg tctaagggat cggttctcca
40642DNAHomo sapiens 6aatgttgccg tgaagatctt cctgagaacc atctgttggg
ta 42738DNAHomo sapiens 7cacgtgtgag acagatgggg gcggttgtga tagacacg
38843DNAHomo sapiens 8aaccatggct agaggattgg cctttcacct acacatccag
ctg 43938DNAHomo sapiens 9caccatcgag ctcgtgaagg agcccttgtc atggaagg
381038DNAHomo sapiens 10cttgaagaag agacccgatc ttctgcacgc tccaccac
381139DNAHomo sapiens 11ttcaccacca cggccgagct ctccttctgc atcctgtcg
391239DNAHomo sapiens 12agccacatcg ctcagacacc gtactcagcg ccagcatcg
39
* * * * *
References