U.S. patent application number 17/552743 was filed with the patent office on 2022-06-02 for rpe cell populations and methods of generating same.
The applicant listed for this patent is CELL CURE NEUROSCIENCES LTD.. Invention is credited to Osnat Bohana-Kashtan, Lior Ann Rosenberg Belmaker, Ofer Wiser.
Application Number | 20220169982 17/552743 |
Document ID | / |
Family ID | 1000006149770 |
Filed Date | 2022-06-02 |
United States Patent
Application |
20220169982 |
Kind Code |
A1 |
Bohana-Kashtan; Osnat ; et
al. |
June 2, 2022 |
RPE CELL POPULATIONS AND METHODS OF GENERATING SAME
Abstract
A population of human polygonal RPE cells is disclosed. At least
95% of the cells thereof co-express premelanosome protein (PMEL17)
and cellular retinaldehyde binding protein (CRALBP), wherein the
trans-epithelial electrical resistance of the cells is greater than
100 ohms. Methods of generating same are also disclosed.
Inventors: |
Bohana-Kashtan; Osnat;
(Tel-Mond, IL) ; Rosenberg Belmaker; Lior Ann;
(Shoham, IL) ; Wiser; Ofer; (Jerusalem,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CELL CURE NEUROSCIENCES LTD. |
Jerusalem |
|
IL |
|
|
Family ID: |
1000006149770 |
Appl. No.: |
17/552743 |
Filed: |
December 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15539473 |
Jun 23, 2017 |
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PCT/IL2015/051269 |
Dec 30, 2015 |
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17552743 |
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62195309 |
Jul 22, 2015 |
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62116972 |
Feb 17, 2015 |
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62097753 |
Dec 30, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/115 20130101;
C12N 2500/38 20130101; C12N 2500/02 20130101; A61K 35/30 20130101;
C12N 5/0621 20130101; C12N 2533/52 20130101; C12N 2506/02 20130101;
C12N 2501/15 20130101; C12N 2501/16 20130101 |
International
Class: |
C12N 5/079 20060101
C12N005/079; A61K 35/30 20060101 A61K035/30 |
Claims
1. (canceled)
2. A method of treating age-related macular degeneration in a
subject comprising: administering into the subretina of the
subject, a composition comprising human retinal pigment epithelium
(RPE) cells, wherein at least 95% of the cells co-express
premelanosome protein (PMEL17) and cellular retinaldehyde binding
protein (CRALBP), wherein the cells have a trans-epithelial
electrical resistance (TEER) greater than 100 ohms and, thereby
treating the subject.
3. The method of claim 2, wherein the purity of the RPE cells is
greater than 98%.
4. The method of claim 2, wherein the PMEL17 and CRABP
co-expression in the RPE cells are increased by at least two fold,
at least 3 fold, at least 4 fold, at least 5 fold, at least 10
fold, at least 20 fold, at least 30 fold, at least 40 fold, or at
least 50 as compared to control cells.
5. The method of claim 2, wherein the cells have a TEER greater
than 300 ohms, greater than 400 ohms, greater than 500 ohms, or
greater than 600 ohms.
6. The method of claim 2, wherein the cells secrete pigment
epithelium-derived factor (PEDF) and vascular endothelial growth
factor (VEGF) in a polarized manner, and wherein the ratio of
apical/basal secretion of PEDF or VEGF is greater than 1.
7. The method of claim 6, wherein the ratio of apical/basal
secretion of PEDF is between 1 and 9.
8. The method of claim 6, wherein the ratio of apical/basal
secretion of VEGF is between 1 and 3.
9. The method of claim 6, wherein the secretion of PEDF and VEGF
remains stable following incubation of the cells at 2-8.degree. C.
for 6 hours, 8 hours, 10 hours, 12 hours or 24 hours.
10. The method of claim 2, wherein the TEER of the cells remains
stable in the cells following their incubation at 2-8.degree. C.
for 6 hours, 8 hours, 10 hours, 12 hours or 24 hours.
11. The method of claim 2, wherein the number of RPE cells
increases after administration to the subretina of the subject.
12. The method of claim 2 wherein the composition is administered
in an amount from about 50.times.103 cells to about 100.times.103
cells per administration.
13. The method of claim 2, wherein the cells secrete angiogenin,
tissue inhibitor metalloproteinase 2 (TIMP 2), soluble glycoprotein
130 (sgp 130) and soluble form of the ubiquitous membrane receptor
1 for tumor necrosis factor-.alpha. (sTNF-R1) in a polarized
manner.
14. The method of claim 2, wherein the cells are capable of
rescuing visual acuity.
15. The method of claim 2, wherein the cells are generated by
ex-vivo differentiation of human embryonic stem cells.
16. The method of claim 2, wherein the RPE cells are generated by:
(a) culturing human embryonic stem cells or induced pluripotent
stem cells in a medium comprising nicotinamide, wherein said medium
is devoid of activin A; (b) culturing the differentiating cells in
a medium comprising nicotinamide and activing A to generate cells
which are further differentiated towards the RPE lineage; and (c)
culturing the further differentiated cells in a medium comprising
nicotinamide, and wherein said medium is devoid of activin A.
17. The method of claim 16, wherein said embryonic stem cells or
induced pluripotent stem cells are propagated in a medium
comprising bFGF and TGF.beta..
18. The method of claim 2, wherein the RPE cells comprise a
polygonal shape morphology.
19. The method of claim 2, wherein the RPE cells comprise less than
1:250,000 of Oct4+TRA-1-60+ cells.
20. The method of claim 2, wherein at least 80% of the RPE cells
express Bestrophin 1, PAX-6 or microphthalmia-associated
transcription factor (MITF).
21. A method of treating a retinal disease in a subject comprising:
administering into the subretina of the subject, a composition
comprising human retinal pigment epithelium (RPE) cells, wherein at
least 95% of the cells co-express premelanosome protein (PMEL17)
and cellular retinaldehyde binding protein (CRALBP), wherein the
cells have a trans-epithelial electrical resistance greater than
100 ohms.
22. The method of claim 21, wherein the retinal disease comprises
age related macular degeneration (AMD), retinitis pigmentosa (RP),
retinoschisis, lattice degeneration, or Best disease.
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] The present invention, in some embodiments thereof, relates
to retinal pigment epithelium cells and, more particularly, but not
exclusively, to assessment of such cells as a therapeutic. The
present invention also relates to generation of retinal pigment
epithelium cells from embryonic stem cells.
[0002] The retinal pigment epithelium (RPE) is a monolayer of
pigmented cells, which lies between the neural retina and the
choriocapillaris. The RPE cells play crucial roles in the
maintenance and function of the retina and its photoreceptors.
These include the formation of the blood-retinal barrier,
absorption of stray light, supply of nutrients to the neural
retina, regeneration of visual pigment, and uptake and recycling of
shed outer segments of photoreceptors.
[0003] Retinal tissue may degenerate for a number of reasons. Among
them are: artery or vein occlusion, diabetic retinopathy and
retinopathy of prematurity, which are usually hereditary. Diseases
such as retinitis pigmentosa, retinoschisis, lattice degeneration,
Best disease, and age related macular degeneration (AMD) are
characterized by progressive types of retinal degeneration.
[0004] RPE cells may potentially be used for cell replacement
therapy of the degenerating RPE in retinal diseases mentioned
above. It may be also used as a vehicle for the introduction of
genes for the treatment of retinal degeneration diseases. These
cells may also serve as an in vitro model of retinal degeneration
diseases, as a tool for high throughput screening for a therapeutic
effect of small molecules, and for the discovery and testing of new
drugs for retinal degeneration diseases. RPE cells could also be
used for basic research of RPE development, maturation,
characteristics, properties, metabolism, immunogenicity, function
and interaction with other cell types.
[0005] Human fetal and adult RPE has been used as an alternative
donor source for allogeneic transplantation. However, practical
problems in obtaining sufficient tissue supply and the ethical
concerns regarding the use of tissues from aborted fetuses limit
widespread use of these donor sources. Given these limitations in
supply of adult and fetal RPE grafts, the potential of alternative
donor sources have been studied. Human pluripotent stem cells
provide significant advantages as a source of RPE cells for
transplantation. Their pluripotent developmental potential may
enable their differentiation into authentic functional RPE cells,
and given their potential for infinite self renewal, they may serve
as an unlimited donor source of RPE cells. Indeed, it has been
demonstrated that human embryonic stem cells (hESCs) and human
induced pluripotent stem cells (iPS) differentiate into RPE cells
in vitro, attenuate retinal degeneration and preserve visual
function after subretinal transplantation to the Royal College of
Surgeons (RCS) rat model of retinal degeneration that is caused by
RPE dysfunction. Therefore, pluripotent stem cells may be an
unlimited source for the production of RPE cells.
[0006] Current protocols for the derivation of RPE cells from
pluripotent stem cells yields mixed populations of pigmented and
non-pigmented cells. However, pure populations of pigmented cells
are desired for the usage of RPE cells in basic research, drug
discovery and cell therapy.
[0007] Background art includes WO 2013/114360, WO 2008/129554 and
WO 2013/184809.
SUMMARY OF THE INVENTION
[0008] According to an aspect of some embodiments of the present
invention there is provided a population of human polygonal RPE
cells, wherein at least 95% of the cells thereof co-express
premelanosome protein (PMEL17) and cellular retinaldehyde binding
protein (CRALBP), wherein the trans-epithelial electrical
resistance of the population of cells is greater than 100 ohms.
[0009] According to an aspect of some embodiments of the present
invention there is provided a population of human RPE cells,
wherein at least 80% of the cells thereof co-express premelanosome
protein (PMEL17) and cellular retinaldehyde binding protein
(CRALBP) and wherein cells of the population secrete each of
angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2),
soluble glycoprotein 130 (sgp130) and soluble form of the
ubiquitous membrane receptor 1 for tumor necrosis factor-.alpha.
(sTNF-R1).
[0010] According to embodiments of the invention, the cells of the
population secrete each of angiogenin, tissue inhibitor of
metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) and
soluble form of the ubiquitous membrane receptor 1 for tumor
necrosis factor-.alpha. (sTNF-R1).
[0011] According to embodiments of the invention, the cells secrete
the angiogenin, the TIMP2, the sgp130 or the sTNF-R1 in a polarized
manner.
[0012] According to embodiments of the invention, the cells secrete
each of the angiogenin, the TIMP2, the sgp130 and the sTNF-R1 in a
polarized manner.
[0013] According to embodiments of the invention, the ratio of
apical secretion of sgp130: basal secretion of sgp130 is greater
than 1.
[0014] According to embodiments of the invention, the ratio of
apical secretion of sTNF-R1: basal secretion of sTNF-R1 is greater
than 1.
[0015] According to embodiments of the invention, the ratio of
basal secretion of angiogenin: apical secretion of angiogenin is
greater than 1.
[0016] According to embodiments of the invention, the ratio of
apical secretion of TIMP2: basal secretion of TIMP2 is greater than
1.
[0017] According to embodiments of the invention, the number of
Oct4.sup.+TRA-1-60.sup.+ cells in the population is below
1:250,000.
[0018] According to embodiments of the invention, at least 80% of
the cells express Bestrophin 1, as measured by immunostaining.
[0019] According to embodiments of the invention, at least 80% of
the cells express Microphthalmia-associated transcription factor
(MITF), as measured by immunostaining.
[0020] According to embodiments of the invention, more than 50% of
the cells express paired box gene 6 (PAX-6) as measured by
FACS.
[0021] According to embodiments of the invention, the cells secrete
greater than 750 ng of Pigment epithelium-derived factor (PEDF) per
ml per day.
[0022] According to embodiments of the invention, the cells secrete
PEDF and vascular endothelial growth factor (VEGF) in a polarized
manner.
[0023] According to embodiments of the invention, the ratio of
apical secretion of PEDF: basal secretion of PEDF is greater than
1.
[0024] According to embodiments of the invention, the ratio remains
greater than 1 following incubation for 8 hours at 2-8.degree.
C.
[0025] According to embodiments of the invention, the
trans-epithelial electrical resistance of the population of cells
is greater than 100 ohms.
[0026] According to embodiments of the invention, the
trans-epithelial electrical resistance of the cells remains greater
than 100 ohms following incubation for 8 hours at 2-8.degree.
C.
[0027] According to embodiments of the invention, the ratio of
basal secretion of VEGF: apical secretion of VEGF is greater than
1.
[0028] According to embodiments of the invention, the ratio remains
greater than 1 following incubation for 8 hours at 2-8.degree.
C.
[0029] According to embodiments of the invention, the cell
population is capable of rescuing visual acuity in the RCS rat
following subretinal administration.
[0030] According to embodiments of the invention, the cell
population is capable of rescuing photoreceptors for at least 180
days post-subretinal administration in the RCS rat.
[0031] According to embodiments of the invention, the cell
population is generated by ex-vivo differentiation of human
embryonic stem cells.
[0032] According to embodiments of the invention, the cell
population is generated by:
[0033] (a) culturing human embryonic stem cells in a medium
comprising nicotinamide so as to generate differentiating cells,
wherein the medium is devoid of activin A;
[0034] (b) culturing the differentiating cells in a medium
comprising nicotinamide and activin A to generate cells which are
further differentiated towards the RPE lineage; and
[0035] (c) culturing the cells which are further differentiated
towards the RPE lineage in a medium comprising nicotinamide,
wherein the medium is devoid of activin A.
[0036] According to embodiments of the invention, the embryonic
stem cells are propagated in a medium comprising bFGF and
TGF.beta..
[0037] According to embodiments of the invention, the embryonic
stem cells are cultured on human cord fibroblasts.
[0038] According to embodiments of the invention, the steps (a)-(c)
are effected under conditions wherein the atmospheric oxygen level
is less than about 10%.
[0039] According to embodiments of the invention, the method
further comprises culturing the differentiated cells in a medium
under conditions wherein the atmospheric oxygen level is greater
than about 10% in the presence of nicotinamide following step
(c).
[0040] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
the cell population described herein, as the active agent and a
pharmaceutically acceptable carrier.
[0041] According to an aspect of some embodiments of the present
invention there is provided a use of the cell population described
herein, for treating a retinal degeneration.
[0042] According to an aspect of some embodiments of the present
invention there is provided a method of generating RPE cells
comprising:
[0043] (a) culturing pluripotent stem cells in a medium comprising
a differentiating agent so as to generate differentiating cells,
wherein the medium is devoid of a member of the transforming growth
factor .beta. (TGF .beta.) superfamily;
[0044] (b) culturing the differentiating cells in a medium
comprising the member of the transforming growth factor .beta. (TGF
.beta.) superfamily and the differentiating agent to generate cells
which are further differentiated towards the RPE lineage; (c)
culturing the cells which are further differentiated towards the
RPE lineage in a medium comprising a differentiating agent so as to
generate RPE cells, wherein the medium is devoid of a member of the
transforming growth factor .beta. (TGF .beta.) superfamily, wherein
steps (a)-(c) are effected under conditions wherein the atmospheric
oxygen level is less than about 10%.
[0045] According to embodiments of the invention, step (a) is
effected under non-adherent conditions.
[0046] According to embodiments of the invention, the non-adherent
conditions comprise a non-adherent culture plate.
[0047] According to embodiments of the invention, the step (a)
comprises:
[0048] i) culturing the cultured population of human pluripotent
stem cells in a medium comprising nicotinamide, in the absence of
activin A; under non-adherent conditions to generate a cluster of
cells comprising differentiating cells; and subsequently;
[0049] ii) culturing the differentiating cells of (i) in a medium
comprising nicotinamide, in the absence of activin A under adherent
conditions.
[0050] According to embodiments of the invention, the method
further comprises dissociating the cluster of cells prior to step
(ii) to generate clumps of cells or a single cell suspension of
cells.
[0051] According to embodiments of the invention, the method
further comprises culturing the differentiated cells in a medium
under conditions wherein the atmospheric oxygen level is greater
than about 10% in the presence of a differentiating agent following
step (c).
[0052] According to embodiments of the invention, the member of the
transforming growth factor .beta. (TGF .beta.) superfamily is
selected from the group consisting of TGF.beta.1, TGF.beta.3 and
activin A.
[0053] According to embodiments of the invention, the
differentiating agent of step (a) and the differentiating agent of
step (c) are identical.
[0054] According to embodiments of the invention, the
differentiating agent of step (a) is nicotinamide (NA) or
3-aminobenzamide.
[0055] According to embodiments of the invention, the method
further comprises selecting polygonal cells following step (c).
[0056] According to embodiments of the invention, the method
further comprises propagating the polygonal cells.
[0057] According to embodiments of the invention, the propagating
is effected on an adherent surface or an extracellular matrix.
[0058] According to embodiments of the invention, the pluripotent
stem cells comprise embryonic stem cells.
[0059] According to embodiments of the invention, the embryonic
stem cells are propagated in a medium comprising bFGF and TGF$.
[0060] According to embodiments of the invention, the embryonic
stem cells are cultured on human cord fibroblasts.
[0061] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0062] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0063] In the drawings:
[0064] FIG. 1 is a graph illustrating the linearity of the
data.
[0065] FIG. 2 is FACS analysis of negative control hESC cells
stained with anti CRALBP and anti PMEL 17.
[0066] FIG. 3 is FACS analysis of positive control of the reference
RPE line OpRegen.RTM. 5C cells stained with anti CRALBP and anti
PMEL 17.
[0067] FIG. 4 is FACS analysis of 25% Spiked OpRegen.RTM. 5C in
hESCs stained with anti CRALBP and anti PMEL 17.
[0068] FIG. 5 is FACS analysis of 50% Spiked OpRegen.RTM. 5C in
hESCs stained with anti CRALBP and anti PMEL17.
[0069] FIG. 6 is FACS analysis of 75% Spiked OpRegen.RTM. 5C in
hESCs stained with anti CRALBP and anti PMELI7.
[0070] FIG. 7 is FACS analysis of 95% Spiked OpRegen.RTM. 5C in
hESCs stained with anti CRALBP and anti PMEL 17.
[0071] FIG. 8 is FACS analysis of hESCs stained with Isotype
Controls.
[0072] FIG. 9 is FACS analysis of OpRegen.RTM. 5C cells stained
with the Isotype Controls.
[0073] FIG. 10: Co-immunostaining with PMEL17 differentiate RPE
cells (CRALBP+PMEL17+) from non RPE pigmented cells
(PMEL17+CRALBP-; such as melanocytes).
[0074] FIG. 11: Morphology results for Mock 4 and 5 at In Process
Control (IPC) points 5, and 8-10.
[0075] FIG. 12: Manufacturing Process, Steps 1-3: Generation of
Human Cord Fibroblast Feeder Working Cell Bank.
[0076] FIG. 13: Manufacturing Process, Steps 4-5: Expansion of
hESCs.
[0077] FIG. 14: Manufacturing Process, Steps 6-13: Differentiation
into RPE (OpRegen.RTM.) cells.
[0078] FIG. 15: Manufacturing Process, Steps 14-17: Expansion of
pigmented cells.
[0079] FIG. 16: Detailed OpRegen.RTM. manufacturing process and in
process control points (yellow stars, IPCs 1-11). (NUTSPlus,
Nutristem medium containing bFGF and TGF.beta.; NUTSMinus,
Nutristem medium w/o bFGF and TGF.beta.; NIC, Nicotinamide; SBs,
Spheroid bodies).
[0080] FIG. 17: Level of CRALBP+PMEL17+RPE cells along OpRegen.RTM.
Mock production runs 4 and 5. Density plots of IPC points 8 and 11
(*IPC point 8 was tested post cryopreservation) and representative
density plots of positive control OpRegen.RTM. 5C and negative
control HAD-C102 hESCs (range of % CRALBP+PMEL17+ in negative
control was 0.02-0.17%). Numbers within each plot indicate percent
CRALBP+PMEL17+ cells out of the live single cell gated population.
Analysis was done using the FCS express 4 software.
[0081] FIG. 18: Immunofluorescence staining of Mock 5 IPC points 7,
10 and 11 with antibodies specific for the RPE markers Bestrophin
1, MITF, ZO-1 and CRALBP.
[0082] FIGS. 19A-C: Representative color fundus photograph of group
2 (BSS+; FIG. 19A), group 5 contra lateral untreated eyes (OD; FIG.
19B) and group 5 treated eyes (OS; FIG. 19C) at P60. The hyper and
hypo-pigmented areas in the high dose treated eyes (OS) are
presumed to be indicative of transplanted cells.
[0083] FIG. 20: Optokinetic tracking acuity thresholds measured at
P60, P100, P150, and P200. Cell treated groups (group 3-25,000,
group 4-100,000 and group 5-200,000) outperformed all controls with
the group 4 (100,000) and 5 (200,000) dose achieving the best
rescue. Contralateral unoperated eyes were equivalent to group 1
(untreated) and group 2 (vehicle control/BSS+) (not shown).
[0084] FIGS. 21A-B: Graphs illustrating the Focal (FIG. 21A) and
Full field (FIG. 21B) results for a representative rat.
[0085] FIGS. 22A-B: FIG. 22A illustrates a photomontage of
individual images of cresyl violet stained sections of a
representative cell treated eye. Between the arrows illustrates the
location of photoreceptor protection and presumed location of the
grafted cells. FIG. 22B illustrates the comparison between
BSS+(Group 2) injected eyes and representative cell injected eyes
(multiple dosage groups represented) at post-natal day 60, 100, 150
and 200. GCL: Ganglion Cell Layer; ONL: Outer Nuclear Layer; RPE:
Retinal Pigmented Epithelium.
[0086] FIG. 23: Outer nuclear layer thickness measured in number of
nuclei. Each dot represents the count from each animal from every
dose group for all ages.
[0087] FIG. 24: Immunofluorescent images of positive control tissue
and representative experimental cell treated animals at P60, P100,
P150, and P200 stained with anti-human nuclei marker (H.N.M,
green), anti-pre-melanosomal marker (PMEL17, red), anti-human
proliferation marker (Ki67, red), and anti-rat cone arrestin (red).
Dapi (blue) is used for background staining to highlight nuclear
layers. Human melanoma was used as positive control tissue for
PMEL17, human tonsil for Ki67, and juvenile RCS rat retina for cone
arrestin. Downward arrows indicate outer nuclear layer; upward
arrows indicate positively stained human RPE cells (OpRegen.RTM.),
generated as described herein.
[0088] FIG. 25 is a graph illustrating cone quantification
following subretinal transplantation of OpRegen.RTM. cells into the
RCS rat. Cell treated eyes were significantly higher than control
eyes at all ages.
[0089] FIGS. 26A-J: Immunofluorescent staining of OpRegen.RTM.
cells in the subretinal space. FIG. 26A represents an area of
retina with a number of RPE cells (red, arrows) central and no
debris zone (viewed using anti-rat rhodopsin antibody, green;
arrow), but where the cells are not (peripheral), the debris zone
reconstitutes. At higher magnification (FIG. 26B), some rhodopsin
stained outer segments rest along the grafted cells. In addition,
the debris zone reconstitutes as distance from transplanted cells
increases. FIGS. 26C-J are individual slices through the section
showing rhodopsin positive tissue within the transplanted cells
(arrows).
[0090] FIGS. 27A-C are photographs illustrating the biodistribution
of the cells following subretinal injection into NOD-SCID. FIG. 27A
illustrates the ability of OpRegen.RTM. cells to engraft in the
NOD-SCID subretinal space 9 months post transplant. Pigmented cells
stain positive for Human Nuclei and PMEL17. FIG. 27B is a
photograph illustrating the clustered cells at the place of the
bleb following injection.
[0091] FIG. 27C is a photograph illustrating the subsequent
spreading of the cells into a monolayer following injection.
[0092] FIG. 28 is a pictorial illustration of a transwell assay
that may be used to assay the potency of RPE cells.
[0093] FIG. 29 is the results of the FACS analysis illustrating
PAX6 expression in RPE cells generated as described herein (P2-DP,
drug product: Mock IV, Mock V, OpRegen.RTM. batch 2A; HuRPE: normal
human RPE from ScienCell) and along production (PO).
[0094] FIG. 30 is a graph illustrating PAX6 expression in
OpRegen.RTM. cells, as assayed by FACS (HES, human embryonic stem
cells used as negative control).
[0095] FIG. 31 is the results of the FACS analysis illustrating
double staining of PAX6 and CRALBP.
[0096] FIGS. 32A-C are graphs illustrating ELISA assessment of
Angiogenin secretion by OpRegena cells. A. Increased secretion of
angiogenin along Mock V production. B. Secretion of angiogenin by
three different batches of OpRegena cells (Passage 3) and on a
transwell for 3 weeks (Passage 4) during which apical and basal
secretion was assessed. C. Secretion of angiogenin by RPE 7 cells
(Passage 3).
[0097] FIGS. 33A-E illustrate TIMP-1 and TIMP-2 Secretion by
OpRegen.RTM. cells. A. Relative TIMP-1 and TIMP-2 protein levels
detected by protein array. B. ELISA TIMP-2 levels in Mock V
production QC points 3 and 4. C-D. ELISA TIMP-2 secretion levels by
different batches of OpRegen.RTM. cells (Passage 3) and on a
transwell for 3 weeks during which apical and basal secretion was
assessed (Passage 4). E. TIMP-2 levels secreted from RPE 7 and
HuRPE control cells (Passage 3, Days 4 & 14).
[0098] FIGS. 34A-D illustrate sgp130 Secretion by OpRegen.RTM.
Cells as measured by ELISA. A. sgp130 secretion levels in Mock V
production QC points 3 and 4. B-C. Levels of secreted sgp130 by
various batches of OpRegen.RTM. cells (Passage 3) and on a
transwell for 3 weeks during which apical and basal secretion was
assessed (Passage 4). D. sgp130 levels secreted from RPE 7 and
HuRPE control cells (Passage 3, Days 4 & 14).
[0099] FIGS. 35A-D illustrate sTNF-R1 protein levels in
OpRegen.RTM. cell supernatant as measured by ELISA. A. sTNF-R1
levels in cell supernatant from Mock V production QC points 3 and
4. B-C. Levels of sTNF-R1 in the supernatant of OpRegen.RTM.
batches (Passage 3) and on a transwell for 3 weeks during which
apical and basal levels were assessed (Passage 4). D. sTNF-R1
levels in day 4 and day 14 RPE7 and control HuRPE cell cultures
(Passage 3).
[0100] FIG. 36 illustrates the morphology of OpRegen.RTM. 5C
(Reference Line), RPE1 and RPE7 on Transwell. OpRegen.RTM. 5C, RPE1
and RPE7 were imaged weekly (week 1-4) following their seeding on
transwell. OpRegen.RTM. 5C generated a homogeneous polygonal
monolayer from week 1 while RPE1 and RPE7 generated a different
non-homogeneous morphology one week post seeding and holes started
to appear at week 2. RPE1 cells detached from the transwell after 3
weeks in culture.
[0101] FIG. 37 illustrates that RPE1 and RPE7 cells co-express
CRALBP and PMEL-17. FACS Purity assay demonstrated that 99.91% and
96.29% of RPE1 and RPE7 cells, respectively, are double positive
for the RPE markers CRALBP and PMEL-17, similar to the levels seen
in OpRegen.RTM. Mock V cells (Positive Control). HAD-C 102 hESCs
were used as the negative control.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0102] The present invention, in some embodiments thereof, relates
to retinal pigment epithelium cells and, more particularly, but not
exclusively, to assessment of such cells as a therapeutic. The
present invention also relates to generation of retinal pigment
epithelium cells from human embryonic stem cells.
[0103] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0104] The neural retina initiates vision and is supported by the
underlying retinal pigment epithelium (RPE). Dysfunction,
degeneration, and loss of RPE cells are prominent features of Best
disease, subtypes of retinitis pigmentosa (RP), and age-related
macular degeneration (AMD), which is the leading cause of visual
disability in the western world. In these conditions, there is
progressive visual loss that often leads to blindness.
[0105] The retina and adjacent RPE both arise from neural ectoderm.
In lower species, RPE regenerates retina but in mammals,
RPE-mediated regeneration is inhibited and renewal occurs to a very
limited extent via stem cells located at the peripheral retinal
margin.
[0106] Human embryonic stem cells (hESC) may serve as an unlimited
donor source of RPE cells for transplantation. The potential of
mouse, primate, and human ESCs to differentiate into RPE-like
cells, to attenuate retinal degeneration, and to preserve visual
function after subretinal transplantation has been
demonstrated.
[0107] Various protocols for the differentiation of human embryonic
stem cells into RPE cells have been developed (see for example WO
2008/129554).
[0108] The present inventors have now discovered a unique and
simple way of qualifying cell populations which have been
successfully differentiated into RPE cells based on expression of
particular polypeptides. Of the myriad of potential polypeptides
expressed on these differentiated cells, the present inventors have
found that a combination of two particular markers can be used to
substantiate successful differentiation.
[0109] The present inventors have also discovered that secretion of
Pigment epithelium-derived factor (PEDF) may be used as a marker to
substantiate early stages of the RPE differentiation process (see
Table 4).
[0110] Whilst further reducing the present invention to practice,
the present inventors identified additional proteins which are
secreted by RPE cells which may be used, in some embodiments, as a
signature to define the cells.
[0111] Thus, according to one aspect of the present invention there
is provided a method of qualifying whether a cell population is a
suitable therapeutic for treating an eye condition, comprising
analyzing co-expression of premelanosome protein (PMEL 17) and at
least one polypeptide selected from the group consisting of
cellular retinaldehyde binding protein (CRALBP), lecithin retinol
acyltransferase (LRAT) and sex determining region Y-box 9 (SOX 9)
in the population of cells, wherein when the number of cells that
coexpress the PMEL17 and the at least one polypeptide is above a
predetermined level, the cell population is qualified as being a
suitable therapeutic for treating a retinal disorder.
[0112] According to another aspect, there is provided a method of
qualifying whether a cell population is a suitable therapeutic for
treating an eye condition, comprising analyzing co-expression of
cellular retinaldehyde binding protein (CRALBP) and at least one
polypeptide selected from the group consisting of premelanosome
protein (PMEL17), lecithin retinol acyltransferase (LRAT) and sex
determining region Y-box 9 (SOX 9) in the population of cells,
wherein when the number of cells that co-express the CRALBP and the
at least one polypeptide is above a predetermined level, the cell
population is qualified as being a suitable therapeutic for
treating an eye condition.
[0113] As used herein, the phrase "suitable therapeutic" refers to
the suitability of the cell population for treating eye conditions.
Cells which are therapeutic may exert their effect through any one
of a multiple mechanisms. One exemplary mechanism is trophic
supportive effect promoting the survival of degenerating
photoreceptors or other cells within the retina. Therapeutic RPE
cells may also exert their effect through a regeneration mechanism
replenishing mal-functioning and/or degenerating host RPE cells.
According to one embodiment, the RPE cells are mature and have the
functional capability of phagocytosing outer shedded segments of
photoreceptors which include rhodopsin. According to another
embodiment, the RPE cells are not fully mature.
[0114] Eye conditions for which the cell populations serve as
therapeutics include, but are not limited to 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, neo vascular or traumatic injury.
[0115] As mentioned, the method of this aspect of the invention is
carried out by measuring the amount (e.g. percent cells) expressing
premelanosome protein (PMEL17; SwissProt No. P40967) and at least
one polypeptide selected from the group consisting of cellular
retinaldehyde binding protein (CRALBP; SwissProt No. P12271),
lecithin retinol acyltransferase (LRAT; SwissProt No. 095327) and
sex determining region Y-box 9 (SOX 9; P48436).
[0116] Alternatively, the method of this aspect is carried out by
measuring CRALBP (CRALBP; SwissProt No. P12271) and at least one
polypeptide selected from the group consisting of lecithin retinol
acyltransferase (LRAT; SwissProt No. 095327), sex determining
region Y-box 9 (SOX 9; P48436) and PMEL17 (SwissProt No.
P40967).
[0117] Thus, for example, CRALBP and PMEL17 may be measured; PMEL17
and LRAT may be measured, or PMEL17 and SOX9 may be measured.
Alternatively, CRALBP and LRAT may be measured, or CRALBP and SOX9
may be measured.
[0118] It will be appreciated that more than two of the
polypeptides mentioned herein can be measured, for example three of
the above mentioned polypeptides or even all four of the above
mentioned polypeptides.
[0119] Methods for analyzing for expression of the above mentioned
polypeptides typically involve the use of antibodies which
specifically recognize the antigen. Commercially available
antibodies that recognize CRALBP include for example those
manufactured by Abcam (e.g. ab15051 and ab189329, clone B2).
Commercially available antibodies that recognize PMEL17 include for
example those manufactured by Abcam (e.g. ab137062 and ab189330,
clone EPR4864). Commercially available antibodies that recognize
LRAT include for example those manufactured by Millipore (e.g.
MABN644). Commercially available antibodies that recognize SOX9
include for example those manufactured by Abcam (e.g. ab185230).
The analyzing may be carried out using any method known in the art
including flow cytometry, Western Blot, immunocytochemistry,
radioimmunoassay, PCR, etc.
[0120] For flow cytometry, the antibody may be attached to a
fluorescent moiety and analyzed using a fluorescence-activated cell
sorter (FACS). Alternatively, the use of secondary antibodies with
fluorescent moieties is envisioned.
[0121] It will be appreciated that since the polypeptides which are
analyzed are intracellular polypeptides, typically the cells are
permeabilized so that the antibodies are capable of binding to
their targets. Cells may be fixed first to ensure stability of
soluble antigens or antigens with a short half-life. This should
retain the target protein in the original cellular location.
Antibodies may be prepared in permeabilization buffer to ensure the
cells remain permeable. It will be appreciated that when gating on
cell populations, the light scatter profiles of the cells on the
flow cytometer will change considerably after permeabilization and
fixation.
[0122] Methods of permeabilizing the cell membrane are known in the
art and include for example:
[0123] 1. Formaldehyde followed by detergent: Fixation in
formaldehyde (e.g. no more than 4.5% for 10-15 min (this will
stabilize proteins), followed by disruption of membrane by
detergent such as Triton or NP-40 (0.1 to 1% in PBS), Tween 20 (0.1
to 1% in PBS), Saponin, Digitonin and Leucoperm (e.g. 0.5% v/v in
PBS);
[0124] 2. Formaldehyde (e.g. no more than 4.5%) followed by
methanol;
[0125] 3. Methanol followed by detergent (e.g. 80% methanol and
then 0.1% Tween 20);
[0126] 4. Acetone fixation and permeabilization.
[0127] As used herein, the term "flow cytometry" refers to an assay
in which the proportion of a material (e.g. RPE cells comprising a
particular marker) in a sample is determined by labeling the
material (e.g., by binding a labeled antibody to the material),
causing a fluid stream containing the material to pass through a
beam of light, separating the light emitted from the sample into
constituent wavelengths by a series of filters and mirrors, and
detecting the light.
[0128] A multitude of flow cytometers are commercially available
including for e.g. Becton Dickinson FACScan, Navios Flow Cytometer
(Beckman Coulter serial #AT15119 RHE9266 and FACScalibur (BD
Biosciences, Mountain View, Calif.). Antibodies that may be used
for FACS analysis are taught in Schlossman S, Boumell L, et al.,
[Leucocyte Typing V. New York: Oxford University Press; 1995] and
are widely commercially available.
[0129] It will be appreciated that the expression level of the
above mentioned polypeptides may be effected on the RNA level as
well as the protein level. Exemplary methods for determining the
expression of a polypeptide based on the RNA level include but are
not limited to PCR, RT-PCR, Northern Blot etc.
[0130] In order to qualify that the cells are useful as a
therapeutic, the amount of at least two of the polypeptides
co-expressed in the cells should be increased above a statistically
significant level as compared to non-RPE cells (e.g.
non-differentiated embryonic stem cells).
[0131] According to a particular embodiment, in order to qualify
that the cells are useful as a therapeutic, at least 80% of the
cells of the population should express detectable levels of PMEL17
and one of the above mentioned polypeptides (e.g. CRALBP), more
preferably at least 85% of the cells of the population should
express detectable levels of PMEL17 and one of the above mentioned
polypeptides (e.g. CRALBP), more preferably at least 90% of the
cells of the population should express detectable levels of PMEL17
and one of the above mentioned polypeptides (e.g. CRALBP), more
preferably at least 95% of the cells of the population should
express detectable levels of PMEL17 and one of the above mentioned
polypeptides (e.g. CRALBP), more preferably 100% of the cells of
the population should express detectable levels of PMEL17 and one
of the above mentioned polypeptides (e.g. CRALBP as assayed by a
method known to those of skill in the art (e.g. FACS).
[0132] According to another embodiment, in order to qualify that
the cells are useful as a therapeutic, the level of CRALBP and one
of the above mentioned polypeptides (e.g. PMEL17) coexpression
(e.g. as measured by the mean fluorescent intensity) should be
increased by at least two fold, more preferably at least 3 fold,
more preferably at least 4 fold and even more preferably by at
least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold,
at least 40 fold, at least 50 as compared to non-differentiated
ESCs.
[0133] According to a particular embodiment, in order to qualify
that the cells are useful as a therapeutic, at least 80% of the
cells of the population should express detectable levels of CRALBP
and one of the above mentioned polypeptides (e.g. PMEL17), more
preferably at least 85% of the cells of the population should
express detectable levels of CRALBP and one of the above mentioned
polypeptides (e.g. PMEL17), more preferably at least 90% of the
cells of the population should express detectable levels of CRALBP
and one of the above mentioned polypeptides (e.g. PMEL17), more
preferably at least 95% of the cells of the population should
express detectable levels of CRALBP and one of the above mentioned
polypeptides (e.g. PMEL17), more preferably 100% of the cells of
the population should express detectable levels of CRALBP and one
of the above mentioned polypeptides (e.g. PMEL17 as assayed by a
method known to those of skill in the art (e.g. FACS).
[0134] In addition, the cell may be qualified in vivo in animal
models. One such model is the Royal College of Surgeons (RCS) rat
model. Following transplantation, the therapeutic effect of the
cells may be analyzed using methods which include fundus imaging,
optokinetic tracking thresholds (OKT), electroretinogram (ERG),
histology, cone counting and rhodopsin ingestion. These methods are
further described in Example 5, herein below.
[0135] The cells may be qualified or characterized in additional
ways including for example karyotype analysis, morphology, cell
number and viability, potency (barrier function and polarized
secretion of PEDF and VEGF), level of residual hESCs, gram staining
and sterility. Exemplary assays which may be performed are
described in Example 4.
[0136] In addition, the cells may be analyzed for barrier function
and their level of growth factor secretion in a polarized manner
(e.g. Pigment epithelium-derived factor (PEDF) or VEGF, cytokines,
interleukins and/or chemokines).
[0137] For analysis of secreted PEDF, supernatant is collected from
cultures of the cells, and cells are harvested and counted. The
amount of PEDF in the cell's culture supernatants may be quantified
by using a PEDF ELISA assay (such as ELISAquant.TM. PEDF Sandwich
ELISA Antigen Detection Kit, BioProductsMD, PED613) according to
the manufacturer's protocol.
[0138] In addition, the direction of secretion of PEDF and VEGF may
be analyzed in the cells. This may be effected using a transwell
assay as illustrated in FIG. 28. Prior to or following
qualification, the cells may be preserved according to methods
known in the art (e.g. frozen or cryopreserved) or may be directly
administered to the subject.
[0139] The present invention contemplates analyzing cell
populations which comprise retinal pigment epithelial (RPE) cells
from any source. Thus, the cell populations may comprise RPE cells
obtained from a donor (i.e. native RPE cells of the pigmented layer
of the retina) or may comprise RPE cells which were ex-vivo
differentiated from a population of stem cells (hSC-derived RPE
cells, such as pluripotent stem cells--e.g. human embryonic stem
cells). According to another embodiment, the RPE cells are obtained
by transdifferentiation--see for example Zhang et al., Protein Cell
2014, 5(1):48-58, the contents of which are incorporated herein by
reference.
[0140] According to one embodiment, the RPE cells that are analyzed
do not express Pax6.
[0141] According to another embodiment, the RPE cells that are
analyzed express Pax6.
[0142] "Retinal pigment epithelium cells", "RPE cells", "RPEs",
which may be used interchangeably as the context allows, refers to
cells of a cell type functionally similar to that of native RPE
cells which form the pigment epithelium cell layer of the retina
(e.g. upon transplantation within an eye, they exhibit functional
activities similar to those of native RPE cells).
[0143] According to one embodiment, the RPE cell expresses at least
one, two, three, four or five markers of mature RPE cells. Such
markers include, but are not limited to CARLBP, RPE65, PEDF,
PMEL17, Bestrophin and tyrosinase. Optionally, RPE cells may also
express a marker of an RPE progenitor--e.g. MITF. In another
embodiment, the RPE cells express PAX-6. In another embodiment, the
RPE cells express at least one marker of a retinal progenitor cell
including, but not limited to OTX2, SIX3, SIX6 and LHX2.
[0144] According to yet another embodiment, the RPE cells are those
that are differentiated from embryonic stem cells according to the
method described in the Examples section herein below, the contents
of the Examples being as if included in the specification
itself.
[0145] As used herein, the phrase "markers of mature RPE cells"
refers to antigens (e.g. proteins) that are elevated (e.g. at least
2 fold, at least 5 fold, at least 10 fold) in mature RPE cells with
respect to non RPE cells or immature RPE cells.
[0146] As used herein the phrase "markers of RPE progenitor cells"
refers to antigens (e.g. proteins) that are elevated (e.g. at least
2 fold, at least 5 fold, at least 10 fold) in RPE progenitor cells
with respect to non RPE cells.
[0147] According to another embodiment, the RPE cells have a
morphology similar to that of native RPE cells which form the
pigment epithelium cell layer of the retina i.e. pigmented and/or
have a characteristic polygonal shape.
[0148] According to still another embodiment, the RPE cells are
capable of treating diseases such as macular degeneration.
[0149] According to still another embodiment, the RPE cells fulfill
at least 1, 2, 3, 4 or all of the requirements listed herein
above.
[0150] 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 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.
[0151] According to a particular embodiment, the RPE cells are
obtained by directed differentiation of hSCs in the presence of one
or more members of the TGF.beta. superfamily, and exhibit at least
one of the following characteristics: [0152] during
differentiation, the cultured cells respond to TGF.beta. signaling;
[0153] the RPE cells express markers indicative of terminal
differentiation, e.g. bestrophin 1, CRALBP and/or RPE65; [0154]
following transplantation (i.e. in situ), the RPE cells exhibit
trophic effect supporting photoreceptors adjacent to RPE cells;
[0155] 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; [0156] further,
in situ the RPE cells are capable of generating a retinal barrier
and functioning in the visual cycle.
[0157] As used herein, the phrase "stem cells" refers to cells
which are capable of remaining in an undifferentiated state (e.g.,
pluripotent or multipotent stem cells) for extended periods of time
in culture until induced to differentiate into other cell types
having a particular, specialized function (e.g., fully
differentiated cells). Preferably, the phrase "stem cells"
encompasses embryonic stem cells (ESCs), induced pluripotent stem
cells (iPS), adult stem cells, mesenchymal stem cells and
hematopoietic stem cells.
[0158] According to a particular embodiment, the RPE cells are
derived from pluripotent stem cells including human embryonic stem
cells or induced pluripotent stem cells.
[0159] The phrase "embryonic stem cells" refers to embryonic cells
which are capable of differentiating into cells of all three
embryonic germ layers (i.e., endoderm, ectoderm and mesoderm), or
remaining in an undifferentiated state. The phrase "embryonic stem
cells" may comprise cells which are obtained from the embryonic
tissue formed after gestation (e.g., blastocyst) before
implantation of the embryo (i.e., a pre-implantation blastocyst),
extended blastocyst cells (EBCs) which are obtained from a
post-implantation/pre-gastrulation stage blastocyst (see
WO2006/040763) and embryonic germ (EG) cells which are obtained
from the genital tissue of a fetus any time during gestation,
preferably before 10 weeks of gestation. The embryonic stem cells
of some embodiments of the invention can be obtained using
well-known cell-culture methods. For example, human embryonic stem
cells can be isolated from human blastocysts. Human blastocysts are
typically obtained from human in vivo preimplantation embryos or
from in vitro fertilized (IVF) embryos. Alternatively, a single
cell human embryo can be expanded to the blastocyst stage. For the
isolation of human ES cells, the zona pellucida is removed from the
blastocyst and the inner cell mass (ICM) is isolated by surgery, in
which the trophectoderm cells are lysed and removed from the intact
ICM by gentle pipetting. The ICM is then plated in a tissue culture
flask containing the appropriate medium which enables its
outgrowth. Following 9 to 15 days, the ICM derived outgrowth is
dissociated into clumps either by a mechanical dissociation or by
an enzymatic degradation and the cells are then re-plated on a
fresh tissue culture medium. Colonies demonstrating
undifferentiated morphology are individually selected by
micropipette/stem cell tool, mechanically dissected into
fragments/clumps, and re-plated. Resulting ES cells are then
routinely split every 4-7 days. For further details on methods of
preparation human ES cells see Reubinoff et al., Nat Biotechnol
2000, May: 18(5): 559; 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]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].
[0160] It will be appreciated that commercially available stem
cells can also be used according to some embodiments of the
invention. Human ES cells can be purchased from the NIH human
embryonic stem cells registry [Hypertext Transfer
Protocol://grants(dot)nih(dot)gov/stem_cells/registry/current(dot)htm]
and other European registries. Non-limiting examples of
commercially available embryonic stem cell lines are HAD-C102, ESI,
BGO1, BGO2, BGO3, BGO4, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4,
CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2,
HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10,
HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES
18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25,
HUES 26, HUES 27, HUES 28, CyT49, RUES3, WA01, UCSF4, NYUES1,
NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA 1, UCLA 2,
UCLA 3, WA077 (H7), WA09 (H9), WA13 (H13), WA14 (H14), HUES 62,
HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1,
WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES 45, Shef 3, Shef 6,
BJNheml9, BJNhem20, SA001, SA001.
[0161] In addition, ES cells can be obtained from other species as
well, including mouse (Mills and Bradley, 2001), golden hamster
[Doetschman et al., 1988, Dev Biol. 127: 224-7], rat [Tannaccone et
al., 1994, Dev Biol. 163: 288-92] rabbit [Giles et al. 1993, Mol
Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, Mol Reprod
Dev. 1993, 36: 424-33], several domestic animal species [Notarianni
et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994,
Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. 3:
59-67] and non-human primate species (Rhesus monkey and marmoset)
[Thomson et al., 1995, Proc Natl Acad Sci USA. 92: 7844-8; Thomson
et al., 1996, Biol Reprod. 55: 254-9].
[0162] Extended blastocyst cells (EBCs) can be obtained from a
blastocyst of at least nine days post fertilization at a stage
prior to gastrulation. Prior to culturing the blastocyst, the zona
pellucida is digested [for example by Tyrode's acidic solution
(Sigma Aldrich, St Louis, Mo., USA)] so as to expose the inner cell
mass. The blastocysts are then cultured as whole embryos for at
least nine and no more than fourteen days post fertilization (i.e.,
prior to the gastrulation event) in vitro using standard embryonic
stem cell culturing methods.
[0163] Another method for preparing ES cells is described in Chung
et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 Feb. 2008.
This method comprises removing a single cell from an embryo during
an in vitro fertilization process. The embryo is not destroyed in
this process.
[0164] Yet another method for preparing ES cells is by
parthenogenesis. The embryo is also not destroyed in the
process.
[0165] Currently practiced ES culturing methods are mainly based on
the use of feeder cell layers which secrete factors needed for stem
cell proliferation, while at the same time, inhibit their
differentiation. Exemplary feeder layers include Human embryonic
fibroblasts, adult fallopian epithelial cells, primary mouse
embryonic fibroblasts (PMEF), mouse embryonic fibroblasts (MEF),
murine fetal fibroblasts (MFF), human embryonic fibroblast (HEF),
human fibroblasts obtained from the differentiation of human
embryonic stem cells, human fetal muscle cells (HFM), human fetal
skin cells (HFS), human adult skin cells, human foreskin
fibroblasts (HFF), human umbilical cord fibroblasts, human cells
obtained from the umbilical cord or placenta, and human marrow
stromal cells (hMSCs). Growth factors may be added to the medium to
maintain the ESCs in an undifferentiated state. Such growth factors
include bFGF and/or TGF.beta.. In another embodiment, agents may be
added to the medium to maintain the hESCs in a naive
undifferentiated state--see for example Kalkan et al., 2014, Phil.
Trans. R. Soc. B, 369: 20130540.
[0166] Feeder cell free systems have also been used in ES cell
culturing, such systems utilize matrices supplemented with serum
replacement, cytokines and growth factors (including IL6 and
soluble IL6 receptor chimera) as a replacement for the feeder cell
layer. Stem cells can be grown on a solid surface such as an
extracellular matrix (e.g., MatrigelR.TM. or laminin) in the
presence of a culture medium--for example the Lonza L7 system,
mTeSR, StemPro, XFKSR, E8). Unlike feeder-based cultures which
require the simultaneous growth of feeder cells and stem cells and
which may result in mixed cell populations, stem cells grown on
feeder-free systems are easily separated from the surface. The
culture medium used for growing the stem cells contains factors
that effectively inhibit differentiation and promote their growth
such as MEF-conditioned medium and bFGF. However, commonly used
feeder-free culturing systems utilize an animal-based matrix (e.g.,
MatrigelR.TM.) supplemented with mouse or bovine serum, or with MEF
conditioned medium [Xu C, et al. (2001). Feeder-free growth of
undifferentiated human embryonic stem cells. Nat Biotechnol. 19:
971-4] which present the risk of animal pathogen cross-transfer to
the human ES cells, thus compromising future clinical
applications.
[0167] Numerous methods are known for differentiating ESCs towards
the RPE lineage and include both directed differentiation protocols
such as those described in WO 2008/129554, 2013/184809 and
spontaneous differentiation protocols such as those described in
U.S. Pat. No. 8,268,303 and U.S. Patent application 20130196369,
the contents of each being incorporated by reference.
[0168] According to a particular embodiment, the RPE cells are
generated from ESC cells using a directed differentiation
protocol--for example according to that disclosed in the Example
section.
[0169] In one exemplary differentiation protocol, the embryonic
stem cells are differentiated towards the RPE cell lineage using a
first differentiating agent and then further differentiated towards
RPE cells using a member of the transforming growth factor-.beta.
(TGF.beta.) superfamily, (e.g. 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)).
[0170] According to a particular embodiment, the TGF.beta.
superfamily member is selected from the group consisting of
TGF.beta.1, activin A and TGF.beta.3.
[0171] According to a specific embodiment, the member of the
transforming growth factor-.beta. (TGF.beta.) superfamily is
activin A--e.g. between 20-200 ng/ml, e.g. 100-180 ng/ml.
[0172] The first differentiating agent promotes differentiation
towards the RPE lineage. For example, the first differentiating
agent may promote differentiation of the pluripotent stem cells
into neural progenitors. Such cells may express neural precursor
markers such as PAX6.
[0173] According to a particular embodiment, the first
differentiating agent is nicotinamide (NA)--e.g. between 1-100 mM,
5-50 mM, 5-20 mM, e.g. 10 mM.
[0174] 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##
[0175] According to a particular embodiment, the nicotinamide is a
nicotinamide derivative or a nicotinamide mimic. The term
"derivative of nicotinamide (NA)" as used herein denotes a compound
which is a chemically modified derivative of the natural NA. In one
embodiment, the chemical modification may be a substitution of 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. When substituted, one or more hydrogen atoms may be
replaced by a substituent and/or a substituent may be attached to a
N atom to form a tetravalent positively charged nitrogen. Thus, the
nicotinamide of the present invention includes a substituted or
non-substituted nicotinamide. In another embodiment, the chemical
modification may be a deletion or replacement of a single 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).
[0176] 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). Other
exemplary nicotinamide derivatives are disclosed in WO01/55114 and
EP2128244.
[0177] Nicotinamide mimics include modified forms of nicotinamide,
and chemical analogs of nicotinamide which recapitulate the effects
of nicotinamide in the differentiation and maturation of RPE cells
from pluripotent cells. Exemplary nicotinamide mimics include
benzoic acid, 3-aminobenzoic acid, and 6-aminonicotinamide. Another
class of compounds that may act as nicotinamide mimics are
inhibitors of poly(ADP-ribose) polymerase (PARP). Exemplary PARP
inhibitors include 3-aminobenzamide, Iniparib (BSI 201), Olaparib
(AZD-2281), Rucaparib (AG014699, PF-01367338), Veliparib (ABT-888),
CEP 9722, MK 4827, and BMN-673.
[0178] According to a particular embodiment, the differentiation is
effected as follows:
[0179] a) culture of ESCs in a medium comprising a first
differentiating agent (e.g. nicotinamide); and
[0180] b) culture of cells obtained from step a) in a medium
comprising a member of the TGF.beta. superfamily (e.g. activin A)
and the first differentiating agent (e.g. nicotinamide).
[0181] Preferably step (a) is effected in the absence of the member
of the TGF.beta. superfamily.
[0182] The above described protocol may be continued by culturing
the cells obtained in step (b) in a medium comprising the first
differentiating agent (e.g. nicotinamide), but devoid of a member
of the TGF.beta. superfamily (e.g. activin A). This step is
referred to herein as step (c).
[0183] The above described protocol is now described in further
detail, with additional embodiments.
[0184] The differentiation process is started once sufficient
quantities of ESCs are obtained. They are typically removed from
the adherent cell culture (e.g. by using collagenase A, dispase,
TrypLE select, EDTA) and plated onto a non-adherent substrate (e.g.
Hydrocell non-adherent cell culture plate) in the presence of
nicotinamide (and the absence of activin A). Exemplary
concentrations of nicotinamide are between 1-100 mM, 5-50 mM, 5-20
mM, e.g. 10 mM. Once the cells are plated onto the non-adherent
substrate, the cell culture may be referred to as a cell
suspension, preferably free floating clusters in a suspension
culture, i.e. aggregates of cells derived from human embryonic stem
cells (hESCs). The cell clusters do not adhere to any substrate
(e.g. culture plate, carrier). Sources of free floating stem cells
were previously described in WO 06/070370, which is herein
incorporated by reference in its entirety. This stage may be
effected for a minimum of 1 day, more preferably two days, three
days, 1 week or even 10 days. Preferably, the cells are not
cultured for more than 2 weeks in suspension together with the
nicotinamide (and in the absence of the TGF.beta. superfamily
member e.g. activin A).
[0185] According to a preferred embodiment, when the cells are
cultured on the non-adherent substrate, the atmospheric oxygen
conditions are manipulated such that the percentage is equal or
less than about 20%, 15%, 10%, more preferably less than about 9%,
less than about 8%, less than about 7%, less than about 6% and more
preferably about 5% (e.g. between 1%-20%, 1%-10% or 0-5%).
[0186] Examples of non-adherent cell culture plates include those
manufactured by Hydrocell (e.g. Cat No. 174912), Nunc etc.
[0187] Typically, the clusters comprise at least 50-500,000,
50-100,000, 50-50,000, 50-10,000, 50-5000, 50-1000 cells. According
to one embodiment, the cells in the clusters are not organized into
layers and form irregular shapes. In one embodiment, the clusters
are devoid of pluripotent embryonic stem cells. In another
embodiment, the clusters comprise small amounts of pluripotent
embryonic stem cells (e.g. no more than 5%, or no more than 3%
(e.g. 0.01-2.7%) cells that co-express OCT4 and TRA 1-60 at the
protein level). Typically, the clusters comprise cells that have
been partially differentiated under the influence of nicotinamide.
Such cells may express neural precursor markers such as PAX6. The
cells may also express markers of progenitors of other lineages
such as for example alpha-feto protein, MIXL1 and Brachyuri.
[0188] The clusters may be dissociated using enzymatic or
non-enzymatic methods (e.g., mechanical) known in the art.
According to one embodiment, the cells are dissociated such that
they are no longer in clusters--e.g. aggregates or clumps of
2-100,000 cells, 2-50,000 cells, 2-10,000 cells, 2-5000 cells,
2-1000 cells, 2-500 cells, 2-100 cells, 2-50 cells. According to a
particular embodiment, the cells are in a single cell
suspension.
[0189] The cells (e.g. dissociated cells) are then plated on an
adherent substrate and cultured in the presence of nicotinamide
e.g. between 1-100 mM, 5-50 mM, 5-20 mM, e.g. 10 mM (and the
absence of activin A). This stage may be effected for a minimum of
1 day, more preferably two days, three days, 1 week or even 14
days. Preferably, the cells are not cultured for more than 1 week
in the presence of nicotinamide on the adherent cell culture (and
in the absence of activin).
[0190] Altogether, the cells are typically exposed to nicotinamide,
(at concentrations between 1-100 mM, 5-50 mM, 5-20 mM, e.g. 10 mM),
for about 2-3 weeks, and preferably not more than 4 weeks prior to
the addition of the second differentiating factor (e.g. Activin
A).
[0191] Examples of adherent substrates include but are not limited
to collagen, fibronectin, laminin, (e.g. laminin 521).
[0192] Following the first stage of directed differentiation (i.e.
culture in the presence of nicotinamide (e.g. 10 mM) under
non-adherent culture conditions under low oxygen atmospheric
conditions followed by culturing on an adherent substrate in the
presence of nicotinamide under low oxygen atmospheric conditions),
the semi-differentiated cells are then subjected to a further stage
of differentiation on the adherent substrate-culturing in the
presence of nicotinamide (e.g. 10 mM) and activin A (e.g. 20-200
ng/ml, 100-200 ng/ml, e.g. 140 ng/ml, 150 ng/ml, 160 ng/ml or 180
ng/ml). This stage may be effected for 1 day to 10 weeks, 3 days to
10 weeks, 1 week to 10 weeks, one week to eight weeks, one week to
four weeks, for example for at least one week, at least two weeks,
at least three weeks, at least four weeks, at least five weeks, at
least six weeks, at least seven weeks or even eight weeks.
Preferably this stage is effected for about two weeks. According to
one embodiment, this stage of differentiation is also effected at
low atmospheric oxygen conditions--i.e. less than about 20%, 15%,
10%, more preferably less than about 9%, less than about 8%, less
than about 7%, less than about 6% and more preferably about 5%
(e.g. between 1%-20%, 1%-10% or 0-5%).
[0193] Following the second stage of directed differentiation (i.e.
culture in the presence of nicotinamide and activin A on an
adherent substrate), the further differentiated cells may
optionally be subjected to a subsequent stage of differentiation on
the adherent substrate--culturing in the presence of nicotinamide
(e.g. between 1-100 mM, 5-50 mM, 5-20 mM, e.g. 10 mM), in the
absence of activin A. This stage may be effected for at least one
day, 2 days, 3 days, 1 week, at least two weeks, at least three
weeks or even four weeks. Preferably this stage is effected for
about one week. This stage of differentiation may be effected at
low (i.e. less than about 20%, 15%, 10%, more preferably less than
about 9%, less than about 8%, less than about 7%, less than about
6% and more preferably about 5% (e.g. between 1%-20%, 1%-10% or
0-5%) or normal atmospheric oxygen conditions or a combination of
both (i.e. initially at low atmospheric oxygen conditions and
subsequently when lightly pigmented cells are observed, at normal
oxygen conditions).
[0194] According to a particular embodiment, when the atmospheric
oxygen conditions are returned to normal atmospheric conditions the
cells are cultured for at least one more day (e.g. up to two weeks)
in the presence of nicotinamide (e.g. 10 mM) and in the absence of
activin A.
[0195] 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 Nuristem (without bFGF and TGF.beta. for ESC
differentiation, with bFGF and TGF.beta. for ESC expansion)
Neurobasal.TM., KO-DMEM, DMEM, DMEM/F12, Lonza L7 system, mTeSR,
StemPro, XF KSR, E8, 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: [0196] 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; [0197] an extracellular matrix
(ECM) component, such as, without being limited thereto,
fibronectin, laminin, collagen and gelatin. The ECM may them be
used to carry the one or more members of the TGF.beta. superfamily
of growth factors; [0198] an antibacterial agent, such as, without
being limited thereto, penicillin and streptomycin; [0199]
non-essential amino acids (NEAA), neurotrophins which are known to
play a role in promoting the survival of SCs in culture, such as,
without being limited thereto, BDNF, NT3, NT4.
[0200] According to a preferred embodiment, the medium used for
differentiating the ESCs is Nuristem medium (Biological Industries,
05-102-1A or 05-100-1A).
[0201] According to a particular embodiment, differentiation of
ESCs is effected under xeno free conditions.
[0202] According to one embodiment, the proliferation/growth medium
is devoid of xeno contaminants i.e. free of animal derived
components such as serum, animal derived growth factors and
albumin. Thus, according to this embodiment, the culturing is
performed in the absence of xeno contaminants.
[0203] Other methods for culturing ESCs under xeno free conditions
are provided in U.S. Patent Application Publication No.
20130196369, the contents of which are incorporated in their
entirety.
[0204] During differentiation steps, the embryonic stem cells may
be monitored for their differentiation state. Cell differentiation
can be determined upon examination of cell or tissue-specific
markers which are known to be indicative of differentiation.
[0205] Tissue/cell specific markers can be detected using
immunological techniques well known in the art [Thomson J A et al.,
(1998). Science 282: 1145-7]. Examples include, but are not limited
to, flow cytometry for membrane-bound or intracellular markers,
immunohistochemistry for extracellular and intracellular markers
and enzymatic immunoassay, for secreted molecular markers (e.g.
PEDF).
[0206] Thus, according to another aspect of the present invention
there is provided a method of generating retinal epithelial cells
comprising:
[0207] (a) culturing pluripotent stem cells in a medium comprising
a differentiating agent so as to generate differentiating cells,
wherein the medium is devoid of a member of the transforming growth
factor .beta. (TGF .beta.) superfamily;
[0208] (b) culturing the differentiating cells in a medium
comprising the member of the transforming growth factor .beta. (TGF
Q) superfamily and the differentiating agent to generate cells
which are further differentiated towards the RPE lineage;
[0209] (c) analyzing the secretion of Pigment epithelium-derived
factor (PEDF) from the cells which are further differentiated
towards the RPE lineage; and
[0210] (d) culturing the cells which are further differentiated
towards the RPE lineage in a medium comprising a differentiating
agent so as to generate RPE cells, wherein the medium is devoid of
a member of the transforming growth factor .beta. (TGF .beta.)
superfamily, wherein step (d) is effected when the amount of the
PEDF is above a predetermined level.
[0211] Preferably, step (d) is effected when the level of PEDF is
above 100 ng/ml/day, 200 ng/ml/day, 300 ng/ml/day, 400 ng/ml/day,
or 500 ng/ml/day.
[0212] Another method for determining potency of the cells during
or following the differentiation process is by analyzing barrier
function and polarized PEDF and VEGF secretion, as illustrated in
Example 4, herein below.
[0213] Once the cells are promoted into RPE cells, they may be
selected and/or expanded.
[0214] According to a particular embodiment, the selection is based
on a negative selection--i.e. removal of non-RPE cells. This may be
done mechanically by removal of non-pigmented cells or removal of
non-polygonal cells or by use of surface markers.
[0215] According to another embodiment, the selection is based on a
positive selection i.e. selection based on morphology (e.g.
pigmented cells and/or polygonal cells). This may be done by visual
analysis or use of surface markers.
[0216] According to still another embodiment, the selection is
based first on a negative selection and then on a positive
selection.
[0217] Expansion of RPE cells may be effected on an extra cellular
matrix, e.g. gelatin, collagen or poly-D-lysine and laminin. For
expansion, the cells may be cultured in serum-free KOM, serum
comprising medium (e.g. DMEM+20%) or Nuristem medium (06-5102-01-1A
Biological Industries). Optionally, the cells may be exposed to
nicotinamide during the expansion phase--at concentrations between
1-100 mM, 5-50 mM, 5-20 mM, e.g. 10 mM. Under these culture
conditions, the pigmented cells reduce pigmentation and acquire a
fibroid-like morphology. Following further prolonged culture and
proliferation into high-density cultures, the cells re-acquire the
characteristic polygonal shape morphology and preferably also
pigmentation of RPE cells.
[0218] 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.
[0219] The population of RPE cells generated according to the
methods described herein may be characterized according to a number
of different parameters.
[0220] Thus, for example, the RPE cells obtained are polygonal in
shape and are pigmented.
[0221] According to one embodiment, at least 70%, 75%, 80%, 85%
90%, 95%, at least 96%, at least 97%, at least 98%, at least 99% or
even 100% of the cells of the RPE cell populations obtained
co-express both premelanosome protein (PMEL17) and cellular
retinaldehyde binding protein (CRALBP).
[0222] Following administration, the cells described herein are
capable of forming a monolayer (as illustrated in FIG. 27C).
[0223] According to one embodiment, the trans-epithelial electrical
resistance of the cells in a monolayer is greater than 100
ohms.
[0224] Preferably, the trans-epithelial electrical resistance of
the cells is greater than 150, 200, 250, 300, 300, 400, 500, 600,
700, 800 or even greater than 900 ohms.
[0225] According to a particular embodiment, the TEER is between
100-1000 ohms, more preferably between 100-900 ohms for example
between 200-900 ohms, 300-800 ohms, 300-700 ohms, 400-800 ohms or
400-700 ohms.
[0226] Devices for measuring trans-epithelial electrical resistance
(TEER) are known in the art. An exemplary set-up for measuring TEER
is illustrated in FIG. 28.
[0227] It will be appreciated that the cell populations disclosed
herein are devoid of undifferentiated human embryonic stem cells.
According to one embodiment, less than 1:250,000 cells are
Oct4*TRA-1-60' cells, as measured for example by FACS. The cells
also do not express or downregulate expression of GDF3 or TDGF
relative to hESCs as measured by PCR.
[0228] Another way of characterizing the cell populations disclosed
herein is by marker expression. Thus, for example, at least 80%,
85%, or 90% of the cells express Bestrophin 1, as measured by
immunostaining. According to one embodiment, between 90-95% of the
cells express bestrophin.
[0229] According to another embodiment, at least 80%, 85%, 87%, 89%
or 90% of the cells express Microphthalmia-associated transcription
factor (MITF), as measured by immunostaining. For example, between
85-95% of the cells express MITF.
[0230] According to another embodiment, at least 50%, 55%, 60%,
70%, 75% 80% 85%, 87%, 89% or 90% of the cells express paired box
gene 6 (PAX-6) as measured by FACS.
[0231] The cells described herein can also be characterized
according to the quantity and/or type of factors that they secrete.
Thus, according to one embodiment, the cells preferably secrete
more than 500, 750, 1000, or even 2000 ng of Pigment
epithelium-derived factor (PEDF) per ml per day, (e.g. following 14
days in culture) as measured by ELISA.
[0232] It will be appreciated that the RPE cells generated herein
secrete PEDF and vascular endothelial growth factor (VEGF) in a
polarized manner. According to particular embodiments, the ratio of
apical secretion of PEDF: basal secretion of PEDF is greater than
1. According to particular embodiments, the ratio of apical
secretion of PEDF: basal secretion of PEDF is greater than 2.
According to particular embodiments, the ratio of apical secretion
of PEDF: basal secretion of PEDF is greater than 3. In addition,
the ratio of basal secretion of VEGF: apical secretion of VEGF is
greater than 1. According to particular embodiments, the ratio of
basal secretion of VEGF: apical secretion of VEGF is greater than
1.5, 2 or 2.5.
[0233] The cells of the present invention secrete additional
factors including for example angiogenin, the immunomodulatory
factors IL-6, sgp130, MIF, sTNF-R1, sTRAIL-R3, MCP-1 and
Osteoprotegerin, the extracellular matrix regulators TIMP-1 and
TIMP-2 and the protein Ax1.
[0234] According to another aspect, at least 80% of the cells of
the cell population co-express premelanosome protein (PMEL17) and
cellular retinaldehyde binding protein (CRALBP) and further a
portion (at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%)
of the cells secrete/shed each of angiogenin, tissue inhibitor of
metalloproteinase 2 (TIMP 2), soluble glycoprotein 130 (sgp130) and
soluble form of the ubiquitous membrane receptor 1 for tumor
necrosis factor-.alpha. (sTNF-R1).
[0235] It will be appreciated that in some cases all the cells that
co-express premelanosome protein (PMEL17) and cellular
retinaldehyde binding protein (CRALBP) also secrete/shed
angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2),
soluble glycoprotein 130 (sgp130) and soluble form of the
ubiquitous membrane receptor 1 for tumor necrosis factor-.alpha.
(sTNF-R1).
[0236] In other cases the majority (more than 50%, 60%, 70%, 80,
90% of the cells that co-express premelanosome protein (PMEL17) and
cellular retinaldehyde binding protein (CRALBP) also secrete/shed
angiogenin, tissue inhibitor of metalloproteinase 2 (TIMP 2),
soluble glycoprotein 130 (sgp130) and soluble form of the
ubiquitous membrane receptor 1 for tumor necrosis factor-.alpha.
(sTNF-R1).
[0237] The RPE cells generated herein preferably secrete
angiogenin, TIMP2, sgp130 and sTNF-R1 in a polarized manner.
[0238] According to particular embodiments, the ratio of apical
secretion of sgp130: basal secretion of sgp130 is greater than 1.
According to particular embodiments, the ratio of apical secretion
of sgp130: basal secretion of sgp130 is greater than 2. According
to particular embodiments, the ratio of apical secretion of sgp130:
basal secretion of sgp130 is greater than 3.
[0239] Furthermore, the ratio of apical sTNF-R1: basal sTNF-R1 is
greater than 1. According to particular embodiments, the ratio of
apical sTNF-R1: basal sTNF-R1 is greater than 2. According to
particular embodiments, the ratio of apical sTNF-R1: basal sTNF-R1
is greater than 3.
[0240] In addition, the ratio of basal secretion of angiogenin:
apical secretion of angiogenin is greater than 1. According to
particular embodiments, the ratio of basal secretion of angiogenin:
apical secretion of angiogenin is greater than 1.5, 2, 2.5 or
3.
[0241] Furthermore, the ratio of apical secretion of TIMP2: basal
secretion of TIMP2 is greater than 1. According to particular
embodiments, the ratio of apical secretion of TIMP2: basal
secretion of TIMP2 is greater than 2. According to particular
embodiments, the ratio of apical secretion of TIMP2: basal
secretion of TIMP2 is greater than 3.
[0242] The stability of the cells is another characterizing
feature. Thus, for example the amount of PEDF secretion remains
stable in the cells following their incubation at 2-8.degree. C.
for 6 hours, 8 hours, 10 hours, 12 hours or even 24 hours. Further,
the polarized secretion of PEDF and VEGF remains stable following
incubation of the cells at 2-8.degree. C. for 6 hours, 8 hours, 10
hours, 12 hours or even 24 hours. Further, the TEER of the cells
remains stable in the cells following their incubation at
2-8.degree. C. for 6 hours, 8 hours, 10 hours, 12 hours or even 24
hours.
[0243] In another embodiment, the cells are characterized by their
therapeutic effect. Thus, for example the present inventors have
shown that the cell populations are capable of rescuing visual
acuity in the RCS rat following subretinal administration. In
addition, the cell populations are capable of rescuing
photoreceptors (e.g. cone photoreceptors) for up to 180 days (in
some embodiments at least 180 days) post-subretinal administration
in the RCS rat.
[0244] It would be well appreciated by those versed in the art that
the derivation of RPE cells is of great benefit. They may be used
as an in vitro model for the development of new drugs to promote
RPE cell survival, regeneration and function. 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.
[0245] 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.
[0246] 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 and or cell culture systems 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 and or cell
culture systems are well known to those versed in the art.
[0247] Harvesting of the cells may be performed by various methods
known in the art. Non-limiting examples include mechanical
dissection and dissociation with papain or trypsin (e.g. TrypLE
select). Other methods known in the art are also applicable.
[0248] The RPE cells generated as described herein 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 choroid). In addition, dependent upon migratory
ability and/or positive paracrine effects of the cells,
transplantation into additional ocular compartments can be
considered including the inner or outer retina, the retinal
periphery and within the choroids.
[0249] Retinal diseases which may be treated using the RPE cells
described herein include, but are not limited to retinitis
pigmentosa, retinoschisis, lattice degeneration, Best disease, and
age related macular degeneration (AMD).
[0250] 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).
[0251] In accordance with one embodiment, transplantation is
performed via pars plana 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.
[0252] 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.
[0253] Thus, the invention also pertains to pharmaceutical
compositions of RPE cells described herein. The composition is
preferably such suitable for transplantation into the eye. Thus,
for example, the RPE cells may be formulated in an intraocular
irrigating solution such as BSS Plus.TM..
[0254] It is expected that during the life of a patent maturing
from this application many relevant technologies will be developed
for the generation of RPE cells, and the term RPE cells is intended
to include all such new technologies a priori.
[0255] As used herein the term "about" refers to .+-.10%.
[0256] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0257] The term "consisting of" means "including and limited
to".
[0258] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0259] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0260] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0261] As used herein, the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0262] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical
symptoms of a condition or substantially preventing the appearance
of clinical or aesthetical symptoms of a condition.
[0263] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0264] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0265] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
[0266] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Qualification of the CRALBP/PMEL 17 Double Staining F ACS
Method
[0267] The aim of this study was to qualify the CRALBP/PMEL 17
double staining FACS method by demonstrating the method's accuracy
and precision in a minimum of 6 independent spiking assays over at
least 3 testing days. The assay qualification was performed using
OpRegen.RTM. batch 5C as the positive control cells and HAD-C
102-hESCs, as the negative control cells. A calibration curve of
known quantities of RPE (OpRegen.RTM. 5C) spiked into hESCs was
used for testing the accuracy and precision at different spiking
points. The expected accuracy and precision were up to 25% at all
points.
[0268] Staining Protocol: Negative Control hESC cells taken from a
cryopreserved hESC bank (HAD-C 102 p48 4.5.2014) were thawed in
Nutristem (containing HSA) according to sponsor protocols. Positive
Control RPE cell stock: OpRegen.RTM. batch 5C cells (reference
line) were thawed into in 20% HS-DMEM according to sponsor
protocols. Thawed OpRegen.RTM. 5C and HAD-C102 hESC were spun down,
re-suspended in 1 ml PBS (-), filtered through a 35 .mu.M cell
strainer and counted with Trypan Blue. The cell concentration was
adjusted to 0.73.times.10.sup.6-10.sup.6 cells/nil in PBS (-). 1
.mu.l/ml FVS450 was added to each cell suspension followed by
vortexing and incubation for 6 minutes at 37.degree. C. FVS450 was
washed with 0.1% BSA, and re-suspended in 0.1% BSA-Fe-block (5 min
at RT) to block all Fc-epitopes on the cells. Cells were then
washed with PBS (-) and fixed in 80% Methanol (5 min at 4.degree.
C.). Fixed cells were washed once with PBS (-), once with 0.1%
PBS-T, and permeabilized with 0.1% PBS-T (20 minutes at RT).
Permeabilization solution was replaced with 10% Normal goat scrum
(NGS) Blocking Solution (200,000 cells/50 .mu.l) for at least 30
minutes (max one hour) at RT. During incubation time quality sample
tubes (QSs) were prepared and at the end of blocking, cells were
divided and immunostained. Cells were incubated with primary
antibodies for 30 minutes followed by 3 washes with 0.1% PBS-T and
30 min incubation with secondary antibodies and 3 washes with 0.1%
PBS-T.
[0269] Negative and positive control cells were stained with the
viability stain FVS450, fixed, blocked and permeabilized. A
calibration curve of known quantities of positive control RPE
(OpRegen.RTM. 5C) cells in negative control hESCs, at 4
concentrations (25%. 50%, 75%, and 95% RPE in hESC), was then
generated based on the Trypan Blue viability cell count of each
population. Negative and positive control cells and the mixed
populations were immunostained with primary monoclonal antibodies
specific to the RPE markers CRALBP and PMEL 17, followed by
staining with matched secondary antibodies (anti-mouse-FITC and
anti-rabbit-Alexa Fluor 647, respectively). Stained cells were FACS
analyzed to measure the percent viable single cell gated
CRALBP+PMEL17+ cells.
[0270] Results
[0271] Accuracy: Accuracy of the assay was determined from test
results of 4 levels of spiked RPEs (25%, 50%, 75% and 95%). The
accuracy of the RPE stock (OpRegen.RTM.5C) was determined with
respect to it being potentially 100% RPE cells. Each level values
were analyzed by six independent runs/determinations.
[0272] The 50% concentration level was considered to be the lower
limit of quantitation with an expected accuracy of up to 25% (50%
level ranged from -8.41 to 20.14; 75% and 99.5% levels ranged from
-5.32 to 6.88).
[0273] These results meet the expected outcomes for relative bias
of up to 25%. and indicate that the assay is accurate for
determination of CRALBP+PMEL17+ double positive cells in
concentrations ranging from 50-99.5%. Since OpRegen.RTM. 5C yields
99.5% CRALBP+PMEL17+ double positive RPE cells, a relative bias of
less than 25% for a result >99.5% cannot be assured.
TABLE-US-00001 TABLE 1 Assigned Measured Relative Run Concentration
(%) Concentration (%) Bias (%) 1 25 20.88 -16.48 2 31.61 26.44 3
32.20 28.80 4 32.01 28.04 5 25.71 2.84 6 26.87 7.48 1 50% 45.93
-8.14 2 60.08 20.16 3 56.87 13.74 4 58.51 17.02 5 50.56 1.12 6
49.52 -0.96 1 75% 71.01 -5.32 2 79.64 6.19 3 78.41 4.55 4 80.16
6.88 5 73.85 -1.53 6 72.94 -2.75 1 95% 93.94 -1.12 2 96.14 1.20 3
95.11 0.12 4 95.59 1.01 5 93.81 -1.25 6 93.70 -1.37 1 100% 98.79
-1.21 2 99.69 -0.31 3 99.62 -0.38 4 99.59 -0.41 5 99.60 -0.40 6
99.48 -0.52
[0274] Intermediate Precision: The intermediate precision of the
assay was determined from results of 6 assays carried out by one
operator. In each assay the percent single viable RPEs was
determined and from that the % CY was calculated. Table 2
summarizes the test results. As shown, % CY for all concentration
levels was below 20% and can be measured with adequate precision. %
CY for the concentration levels 25%, 50%, 75%, 95% and 100% RPEs,
were 16.14%, 10.61%, 5.10%, 1.17%, and 0.34%, respectively. These
results meet the expected values for precision. The measured
percent RPEs is within 20% of the expected value at all
concentrations. These results indicate that the assay is precise
for determination of RPEs in concentrations ranging from
25-99.5%.
TABLE-US-00002 TABLE 2 Assigned Measured Concentration (%) Run
Concentration (% RPE) 25 1 20.88 2 31.61 3 32.20 4 32.01 5 25.71 6
26.87 Mean % RPE 28.21 SD 4.55 % CV 16.14 50 1 45.93 2 60.08 3
56.87 4 58.51 5 50.56 6 49.52 Mean % RPE 53.58 SD 5.68 % CV 10.61
75 1 71.01 2 79.64 3 78.41 4 80.16 5 73.85 6 72.94 Mean % RPE 76.00
SD 3.88 % CV 5.10 95 1 93.94 2 96.14 3 95.11 4 95.96 5 93.81 6
93.70 Mean % RPE 94.78 SD 1.11 % CV 1.17 100 1 98.79 2 99.69 3
99.62 4 99.59 5 99.60 6 99.48 Mean % RPE 99.46 SD 0.34 % CV
0.34
[0275] Repeatability: Sample repeatability was tested in 3 runs
(#2, #3 and #4) in which duplicate OpRegen.RTM. SC samples were
stained and acquired side by side. The results confirmed that
sample identity obtained within an experiment is repeatable and
consistent across samples.
[0276] Linearity/range: As shown in FIG. 1, linearity was measured
using data that were found to be both accurate and precise. The
coefficient of regression between the target (spiked) and measured
results across the tested assay range (50%-100%) was found to be
0.99. Thus, the range of the method which demonstrates acceptable
accuracy and precision and linearity is the range between 50% and
99.5% RPE cells, which covers the expected range of tested
samples.
[0277] Positive control cells: The provisional level of
CRALBP/PMEL17 double positive cells was set at equal to or greater
than 95%.
[0278] Negative control cells: The provisional level of
CRALBP/PMEL17 double positive cells for hESCs was set at equal to
or less than 2%.
[0279] Stability: The results show that stained samples are stable
at 4.degree. C. also after one and 4 days and accuracy is kept
within expected acceptance criteria, therefore the data acquisition
can be performed within 96 hours of sample preparation.
[0280] Conclusion
[0281] The results presented herein indicate that the disclosed
method is qualified and suitable for its intended use of in vitro
determination of RPE purity in OpRegen.RTM. final product and at
different stages along the production process of OpRegen.RTM., with
Accuracy of Relative Bias of <25% and precision of % CV <20%
in the range of 50%-99.5% RPE cells.
Example 2
Assessing the Level of OpRegen.RTM. Purity
[0282] A FACS based method for assessing the level of human retinal
pigment epithelial cells (RPE) purity as well as non-RPE cellular
impurities in RPE cells was developed. Cellular
retinaldehyde-binding protein (CRALBP), one of the visual cycle
components, was bioinformatically identified as a unique marker for
mature RPE cells. Preliminary studies using CRALBP specific
monoclonal antibody have shown purity of above 98% in RPE cells
generated according to methods described herein. These results were
further supported by immunostaining for PMEL17, a melanosome marker
found in RPE. In addition, different from some RPE specific
markers, CRALBP is not expressed in melanocytes, a possible neural
crest cellular contamination.
[0283] Test Sample and Controls: Human primary melanocytes (ATCC,
PCS-200-013) were used as negative control cells for CRALBP and as
positive control cells for PMEL17, type I transmembrane
glycoprotein enriched in melanosomes (melanin granules).
HADC102-hESCs at P29 (OpRegen.RTM. parental line), were used as
negative control cells for both CRALBP and PMEL17. Clinical grade
OpRegen.RTM. cells (batch 2A), and research grade OpRegen.RTM.
(produced in GMP like Mock production; Mock IV D16) were used as
the tested samples. The cells were generated as described in
Example 3.
[0284] Immunostaining and FACS analysis: cells were thawed and
stained using the Fixable Viability Stain (FVS450) (BD 562247),
fixed with 80% Methanol, immunostained with the primary mouse anti
CRALBP (Clone B2, Abcam ab15051), or its isotype control for mouse
IgG2a (Abcam ab170191) and rabbit anti human PMEL17 (Clone EPR4864,
Abeam ab137062) followed by secondary antibodies goat anti mouse
(Dako F0479) and goat anti rabbit (Jackson 111-606-144),
respectively.
[0285] Acquisition of FACS data was performed using a validated
Navios flow cytometer (Beckman Coulter) and analysis was performed
using FlowJo 7.6.
[0286] Results
[0287] Initial FACS data using anti CRALBP monoclonal antibody and
showed that the purity level of OpRegen.RTM. is above 98%.
Melanocytes which are a possible neural crest cellular contaminant
were found negative for the unique RPE specific marker CRALBP
(1.7%). The parental line HADC102-hESCs were negative to CRALBP
(0.2%), as expected.
[0288] The purity level of OpRegen.RTM. stayed above 98% following
double staining with CRALBP and PMEL17 (FIG. 10). Melanocytes
stained positive for PMEL17, as expected, but were negative for the
double marked population (.about.1%). HADC102-hESCs were negative
stained for CRALBP and PMEL17 (0.07%).
Example 3
Description of Manufacturing Process and Process Controls
[0289] OpRegen.RTM. is manufactured from the xeno-free GMP grade
HAD-C 102 hESC line grown on irradiated xeno-free GMP-grade human
umbilical cord fibroblast feeders. Clinical-grade human fibroblast
feeder cell line (CRD008; MCB) and working cell banks (WCBs) were
produced under Good Manufacturing Practice (GMP) and xeno-free
conditions, appropriately tested, characterized and banked. These
were then used in the derivation of clinical-grade hESC line HAD-C
102 from surplus human blastocysts under GMP and xeno-free
conditions.
[0290] At the initial phase of production hESCs are expanded on
irradiated feeders as colonies. They are then transferred to
suspension culture to initiate differentiation in a directed
manner. Spheroid bodies (SBs) are formed and then plated as an
adherent culture under continued directed differentiation
conditions towards a neural fate and subsequently towards RPE
cells. At the end of the differentiation phase non-pigmented areas
are physically excised and pigmented cells are enzymatically
collected, seeded and expanded. Purified hESC-derived RPE cells
(DS) are harvested at passage 2 and immediately processed to the
DP. Duration of the manufacturing process depends on the hESCs
growth rate (.about.2 months from thawing) and in total usually
spans over 4-5 months.
[0291] Each step of the manufacturing process, including the
in-process quality control (QC) tests is briefly described
below.
[0292] Steps 1-3: Generation of human cord fibroblast feeder
Working Cell Bank (WCB). A vial of human cord feeder Master Cell
Bank (MCB) (CRD008-MCB) at passage 3-4 was thawed, expanded in
Dulbecco's Modified Eagle's Medium (DMEM, SH30081.01, Hyclone)
supplemented with 20% human serum (14-498E, Lonza), irradiated
(Gamma cell, 220 Exel, MDS Nordion 3,500 rads) and cryopreserved at
passages 7-8 to generate the working cell banks (WCBs). Prior to
cryopreservation, samples from the feeder cell cultures were tested
for sterility, mycoplasma and Limulus Amebocyte Lysate (LAL),
morphology, karyotype, cell number, and viability. In addition,
post thawing, their identity to the MCB, their inability to
proliferate and their ability to support un-differentiated
HAD-C102-hESC growth were confirmed. If the WCB passed all QC
testing, the bank was released for expansion of hESCs.
[0293] Production Steps 1-3 are depicted in FIG. 12.
[0294] Steps 4-5: Expansion of hECSs. A single vial of the human
cord fibroblast WCB (either CRD008-WCB8 or CRD008-WCB9) was thawed
and plated in center well plates covered with recombinant human
gelatin (RhG100-001, Fibrogen) at a concentration of 70,000-100,000
cells/ml/plate in DMEM (SH30081.01, Hyclone) supplemented with 20%
human serum (14-498E, Lonza). The cells were incubated over night
at 37.degree. C. 5% CO.sub.2 to allow the fibroblasts to attach.
1-4 days later, a sample from HAD-C102-hESC MCB was thawed and
plated for 6-7 days at 37.degree. C. 5% CO.sub.2 on top of the
feeder cells in Nutristem "Plus" Medium (which is GMP-grade and
xeno-free) that contains the growth factors bFGF and TGF-.beta.
(05-102-1A, Biological Industries, Israel). On day 6-7 hESC culture
was mechanically disrupted (using a sterile tip or a disposable
sterile stem cell tool; 14602 Swemed) and passaged into additional
freshly prepared plates containing feeder cells at a concentration
of 70,000-100,000 cells/plate. This was repeated weekly for several
passages to reach the necessary amount of hESC to initiate
differentiation (FIG. 13, Steps 4-5). Prior to their use, expanded
HAD-C102-hESCs were tested for sterility, mycoplasma, LAL,
karyotype, and identity to the MCB. In addition, their pluripotent
morphological appearance as well as unified expression of
pluripotency markers (TRA-1-60, Oct4, and alkaline phosphatase)
were confirmed (FIG. 2, Step 5). Production Steps 4-5 are depicted
in FIG. 13.
[0295] Steps 6-13: Differentiation into RPE cells. Expanded
HAD-C102-hESCs were enzymatically treated with collagenase (4152,
Worthington) for additional expansion in 6 cm cell culture plates
(FIG. 14, Step 6). Expanded HAD-C102-hESCs were then used in the
derivation of the OpRegen.RTM. DS.
[0296] Differentiation of each OpRegen.RTM. batch was initiated by
mechanical transfer of collagenase A harvested clusters of
HAD-C102-hESCs from Step 6 culture to a feeder-free non-adherent 6
cm Hydrocell culture dishes in the presence of Nutristem "Minus"
Medium (that does not contain the growth factors bFGF and
TGF-.beta.; 06-5102-01-1A Biological Industries, Special Order)
supplemented with 10 mM Nicotinamide (N-5535, Sigma) (FIG. 14, Step
7). The plates were then cultured for up to one week under low
oxygen atmosphere (5%) conditions (37.degree. C., 5% CO.sub.2) to
allow the generation of spheroid bodies. Week old spheroid bodies
in suspension were then collected, dissociated gently by pipetting,
and transferred to human laminin (511, Biolamina)-coated 6-well
plates for an additional week of growth under a low oxygen
atmosphere (5%) in the presence of Nutristem "Minus" Medium
supplemented with 10 mM Nicotinamide (FIG. 14, Step 8). The cells
continued to grow under low oxygen (5%) atmosphere for an
additional up to 4 weeks; two weeks in the presence Nutristem
"Minus" Medium supplemented with 10 mM nicotinamide and 140 ng/ml
Activin A (G-120-14E, Peprotech) (FIG. 14, Step 9), followed by up
to 2 weeks in the presence of Nutristem "Minus" Medium supplemented
with only 10 mM nicotinamide (FIG. 14, Step 10). When areas of
light pigmentation became apparent in patches of polygonal cells,
plates were transferred back to normal oxygen (20%) atmosphere
(37.degree. C., 5% CO.sub.2) and were grown for up to 2 weeks in
the presence of Nutristem "Minus" Medium with 10 mM Nicotinamide
(FIG. 14, Step 11). After up to 2 weeks, expanded polygonal patches
with distinctive pigmentation were apparent within areas of
non-pigmented cells (FIG. 14, Step 12) and remaining pigmented
cells were detached and manually collected following 15 minutes
TrypLE Select (12563-011, Invitrogen) treatment at 37.degree. C.
(FIG. 14, Step 13). Production Steps 6-13 are depicted in FIG.
14.
[0297] Steps 14-17: Expansion of OpRegen.RTM. cells. Pigmented
cells were then transferred to 6-well gelatin-coated plates
(0.5-1.times.10.sup.6 cells/plate; P0) for a 2-3 days of growth in
the presence of DMEM (SH30081.01, Hyclone) supplemented with 20%
human serum (14-498E, Lonza) (FIG. 15, Step 14). DMEM was then
replaced with Nutristem "Minus" Medium and cells were grown for 2-3
weeks until the plate was covered with lightly pigmented polygonal
cells (FIG. 15, Step 14). These P0 cells were then expanded in
gelatin-covered flasks for an additional two passages (P1, P2).
Cells at P0 and at P1 were harvested following TrypLE Select
treatment at 37.degree. C., washed and cultured for 2-3 days on
gelatin-coated flasks in the presence of DMEM supplemented with 20%
human serum. DMEM was replaced with Nutristem "Minus" Medium and
the cells were grown for 2-3 weeks until the plate was covered with
lightly pigmented polygonal cells (FIG. 15, Steps 15-16). Cells at
P2 grown in T175 flasks were then harvested following TrypLE Select
treatment at 37.degree. C., re-suspended in DMEM supplemented with
20% human serum, pooled and counted.
[0298] A sample of growth medium from each batch was taken for
sterility, mycoplasma, and LAL testing. The cells morphology was
observed and documented (FIG. 15, Step 17). Production steps 14-17
are depicted in FIG. 15.
Example 4
Process Control Points
[0299] IPC points are depicted in FIG. 16. The sampling points
chosen to assess hESC impurity and RPE purity along the production
process are described below: IPC point 1: Mechanically expanded
HAD-C 102 hESCs prior to their differentiation that have normal
karyotype. This is the starting material in which the highest level
of hESCs is expected. This point was added to evaluate the maximal
hESC level prior to differentiation.
[0300] IPC point 2: Collagenase expanded HAD-C 102 hESCs prior to
their differentiation. At this stage, some differentiation is
expected, and thereby a reduction in the level of cells expressing
Oct4 and TRA-1-60 as well as in the expression level of GDF3 and
TDGF. This point was added to evaluate hESC impurity during the
phase of non-directed differentiation.
[0301] IPC point 3: Spheroid Bodies produced one week post
induction of hESC differentiation under feeder free conditions in
the presence of Nicotinamide. At this earlier stage of
differentiation, hESC impurity during differentiation is expected
at the maximal level and thereby this assessment is expected to
give an indication for the highest level of safety concern.
[0302] IPC point 4: Cells at the end of Activin A treatment.
Activin A directs the differentiation towards RPE cells. At this
point, a major decrease in hESC impurity and a high increase in
expression of RPE markers are expected. This point was added to
monitor hESC differentiation to RPE.
[0303] IPC points 5-7: Cells at the end of the differentiation
process prior and post separation of the non-pigmented areas (IPC
point 6) from the pigmented areas (IPC point 7). IPC points 5 and 6
are expected to contain cellular impurities, while sample 7
represents the product at the end of the differentiation process
prior to its expansion. Cellular contaminations found in sample 6,
may be found is small quantities in sample 7, and in smaller
quantities in the product.
[0304] IPC point 8: Pigmented cells at P0. Pigmented cells at the
end of the differentiation process that were expanded for 2-3
weeks. These cells represent the product two stages prior to the
end of the production process.
[0305] IPC point 9: Pigmented cells at P1. P0 cells that were
expanded for 2-3 weeks. These cells represent the product one stage
prior to the end of the production process.
[0306] IPC point 10: Pigmented cells at P2 prior to
cryopreservation. P1 cells that were expanded for 2-3 weeks are
harvested and pooled. These cells represent the drug substance (DS)
prior to cryopreservation.
[0307] IPC point 11: Cryopreserved pigmented cells at P2. These
cells represent the drug product (DP). Throughout production, at
all sampling points, cell culture medium was collected for
assessment of pigment epithelium derived factor (PEDF) secretion,
known to be secreted from RPE cells.
[0308] Results
[0309] Quantification of TRA-1-60.sub.+Oct4.sub.+hESCs: The level
of hESCs in the various samples collected along the production
process was determined using a highly sensitive, robust
Oct4/TRA-1-60 double staining FACS method. A week following removal
of feeders and growth factors that supports pluripotent cell growth
(TGF.beta. and bFGF), at growth conditions that supports early
neural/eye field differentiation, there were only 0.0106-2.7%
TRA-1-60.sub.+Oct4.sub.+ cells (IPC point 3, Spheroid Bodies).
Following addition of Activin A that promotes RPE differentiation,
the level of TRA-1-60.sub.+Oct4.sub.+ cells was further deceased to
0.00048-0.0168% (IPC point 4, end of activin), and at the end of
differentiation following excision of non-pigmented cells, the
level of TRA-1-60.sub.+Oct4.sub.+ cells was 0.00033-0.03754% (IPC
point 7, pigmented cells). At P0, two stages prior to the end of
the production process, TRA-1-60.sub.+Oct4.sub.+ cells in levels of
0.00009-0.00108% (below LOD-close to LLOQ) were detected (IPC point
8). The levels of TRA-1-60.sub.+Oct4.sub.+ cells at P1 (IPC point
9), P2 prior to cryopreservation (Drug Substance; IPC point 10),
and P2 post cryopreservation (DP; IPC point 11) were below assay
LLOQ (i.e. 0.00004-0.00047%, 0.00000-0.00016% and 0.00000-0.00020%
respectively).
[0310] Relative expression of the pluripotency hESC markers GDF3
and TDGF: The relative expression of the pluripotency genes GDF3
and TDGF at the various IPC points along the production process was
analyzed. There was a gradual reduction in the expression level of
GDF3 and TDGF, which was correlated with the gradual reduction in
the numbers of TRA-1-60.sub.+Oct4.sub.+ cells, along the
differentiation process. At the end of P0, two stages prior to the
end of the production process, P1, and P2 prior (Drug Substance)
and post (Drug Product) cryopreservation, the expression levels of
GDF3 and TDGF were similar to the level of expression seen in the
negative control OpRegen.RTM. 5C cells.
[0311] Quantification of CRALBP+PMEL17.sub.+ cells: Assessment of
CRALBP+PMEL17.sub.+ cells for measurement of RPE purity was
effected at the end of the differentiation phase, at P0 and P2 (IPC
points 8 and 11), respectively), were assessed. As can be seen in
Table 3 and in FIG. 17, the level of CRALBP+PMEL17.sub.+RPE purity
at P0 (IPC point 8), two stages prior to the end of the production
process, was in the range of 98.53-98.83%. Similar level of RPE
purity was detected at P2 post cryopreservation (99.61-99.76%; IPC
point 11) (Table 3).
TABLE-US-00003 TABLE 3 IPC Point Sampling Time and Stage %
CRALBP.sup.+PMEL17.sup.+ Cells IPC Week Stage Mock 4 Mock 5 Range 8
12 Pigmented cells at P0* 98.53 98.83 98.53-98.83 11 18 OpRegen
.RTM. (P2); DP 99.61 99.76 99.61-99.76
[0312] DP, Drug Product. *IPC point 8 was tested post
cryopreservation. Internal assay controls of RPE cells
(OpRegen.RTM. 5C, positive control) spiked into hESCs (HAD-C 102,
negative control) demonstrated accuracy error of <25%.
[0313] Confocal imaging of Bestrophin 1, MITF, and CRALBP
immunostained cells along Mock production runs 4 and 5: Cells were
immunostained for the RPE markers Bestrophin 1, MITF, ZO-1 and
CRALBP at the end of the differentiation phase (IPC point 7), at
the end of the expansion phase (IPC point 10, DS), and post
cryopreservation (IPC point 11, DP). Manually isolated
non-pigmented cells (IPC point 6) were plated for immunostaining,
but during fixation were detached from the plate and thereby could
not be stained. Selected pigmented cells (IPC point 7) plated for
12 days (in mock 5 only, in parallel to cells at P0 from the
ongoing production) and for 28 days were positively stained for all
tested RPE markers and the percent cells expressing Bestrophin 1
and MITF were 93% and 93.3-96.5%, respectively. Similar levels of
Bestrophin 1 and MITF positive cells were detected at P0 (94.9% and
95.9%, respectively; tested only in mock 4), P2 prior
cryopreservation, Drug Substance (92.2-92.75% and 93.7-95.5%,
respectively), and P2 post cryopreservation, Drug Product
(91.1-95.7% and 83.8-94.9%, respectively; decreased MITF
immunostaining in mock 5 demonstrate an outlier of the randomly
selected area for analysis). CRALBP (as well as ZO-1) expression
was detected in all IPC 7, 10 and 11 samples (FIG. 18).
[0314] Relative expression of the RPE markers Bestrophin 1, CRALBP
and RPE65 along Mock productions 2, 4 and 5: The relative
expression of the RPE genes Bestrophin 1, CRALBP and RPE65 at the
various IPC points along the production process was measured. There
was a gradual increase in the relative expression level of
Bestrophin 1, CRALBP and RPE65 along the production process. At the
end of Activin A treatment (IPC point 4), that directs the
differentiation towards RPE cells, the relative levels of
Bestrophin 1, CRALBP and RPE65 were 685, 36, and 325, respectively,
fold higher as compared to their relative levels in mechanically
passaged hESCs prior to differentiation (IPC point 1; mock 4). The
relative expression levels of Bestrophin 1, CRALBP and RPE65
reached a peak from the end of the differentiation stage (IPC
points 5) to the P1 stage (IPC point 9). At these stages the
respective levels of expression were 5,838-11,841, 211-299, and
5,708-8,687, fold higher as compared to the levels in mechanically
passaged hESCs prior to differentiation (IPC point 1).
[0315] Morphology assessment along Mock productions 4 and 5: Cells
were analyzed for morphology at the end of the differentiation
phase (IPC point 5) for estimation of the relative area of
pigmented cells, and at the expansion phases P0-P2 (IPC points
8-10), to verify confluent polygonal morphology. The relative
pigmented cellular area estimated at the end of the differentiation
phase prior to excision of the non-pigmented areas (IPC point 5),
was 32.5%.+-.13.5% (average.+-.SD, n=7 wells of a 6 well plate) in
mock 4 and 60%.+-.13% in mock 5 (average.+-.SD, n=7 wells of a 6
well plate) (see representative images in FIG. 11). Areas of
pigmented cells were selected and expanded. Morphology at the end
of the expansion phases P0 (IPC point 8), P1 (IPC point 9), and P2
(IPC point 10) demonstrated a densely packed culture with a typical
polygonal-shaped epithelial monolayer morphology (FIG. 11).
[0316] PEDF secretion and potency measurement along Mock
productions 4 and 5: Pigment epithelium-derived factor (PEDF),
known to be secreted from RPE cells, was measured in the cell
culture medium at various IPC points along mock productions 4 and
5. As can be seen in Table 4, very low levels of PEDF, in the range
of 4-79 ng/mL/day, were secreted by hESCs (IPC points 1 and 2) and
by spheroid bodies (IPC point 3; end of the first week with
Nicotinamide). At the end of Activin A treatment (IPC point 4),
that directs the differentiation towards RPE cells, the level of
secreted PEDF was in the range of 682-1,038 ng/mL/day, 31-37 fold
higher compared to the level secreted by spheroid bodies. Following
incubation of cells at normal oxygen conditions with Nicotinamide
(IPC point 5), further increase (2.2-4.6 fold) in PEDF secretion to
1,482-4,746 ng/mL/day, was observed. During the expansion phase
(P0-P2, IPCs 8-10, respectively), PEDF secreted levels were in the
range of 2,187-8,681 ng/mL/day, peaking at P0-P1.
TABLE-US-00004 TABLE 4 PEDF secretion along mock productions 4 and
5. PEDF secretion IPC Sampling Time and Stage (ng/mL/day) IPC Week
Stage Mock 4 Mock 5 Range 1 0 Mechanically passaged hESCs 1
Mechanically passaged ND ND NA hESCs 2 Mechanically passaged 4 ND
NA hESCs 2 3 Collagenase passaged 21 79 21-79 hESCs 3 4 Spheroid
Bodies 22 28 22-28 4 7 Cells at the end of Activin 682 1,038
682-1,038 A treatment 5 10 Cells at the end of 1,482 4,746
1,482-4,746 differentiation 8 12 Pigmented cells at P0 7,523 7,951
7,523-7,951 9 14 Pigmented cells at P1 8,681 7,287 7,287-8,681 10
16 OpRegen .RTM. (P2); DS 2,187 5,147 2,187-5,147 11 18 OpRegen
.RTM. (P2); DP 2,462 3,936 2,462-3,936 ND, Not done; NA, Not
Applicable; DS, Drug Substance; DP, Drug Product.
[0317] Tight junctions generated between RPE cells enable the
generation of the blood-retinal barrier and a polarized PEDF and
VEGF secretion. PEDF is secreted to the apical side where it acts
as an anti angiogenic and neurotropic growth factor. VEGF is mainly
secreted to the basal side, where it acts as a proangiogenic growth
factor on the choroidal endothelium. RPE polarization (barrier
function and polarized PEDF and VEGF secretion) was measured in a
transwell system at the end of P0 (IPC point 8), end of P2 prior to
cryopreservation (IPC point 10), and end of P2 post
cryopreservation (IPC point 11). As can be seen in Table 5, barrier
function/trans-epithelial electrical resistance (TEER) and
polarized secretion of PEDF and VEGF were demonstrated at all IPC
points.
TABLE-US-00005 TABLE 5 Polarization Transwell- Transwell-
Transwell- TEER PEDF ratio at VEGF ratio IPC Point Sampling PEDF
Day 14 (.OMEGA.) at Week 3 at Week 3 Time and Stage (ng/mL/day)
Week 3 (Apical/Basal) (Basal/Apical) IPC Week Stage M4 M5 M4 M5 M4
M5 M4 M5 Range 8 12 Pigmented 1,985 3,292 768 933 6.01 6.72 3.01
3.09 PEDF cells D14: at P0 1,985-3,292 TEER: 768-933 PEDF ratio:
6.01-6.72 VEGF ratio: 3.01-3.09 10 16 OpRegen .RTM. 1,754 4,250 819
941 5.72 4.72 2.54 2.73 PEDF (P2); D14: DS 1,754-4,250 TEER:
819-941 PEDF ratio: 4.72-5.72 VEGF ratio: 2.54-2.73 11 18 OpRegen
.RTM. 2,462 3,936 688 616 6.78 3.93 2.57 2.74 PEDF (P2); D14: DP
2,462-3,936 TEER: 616-688 PEDF ratio: 3.93-6.78 VEGF ratio:
2.57-2.74 ND, Not Done; DS, Drug Substance; DP, Drug Product. PEDF
and VEGF were measured by ELISA. PEDF day 14 was collected from the
cells during their culture in a 12-well plate. Cells were then
passaged onto a transwell and cultured for 6 weeks, during which
TEER, and secretion of VEGF and PEDF from the basal and apical
sides of the transwell were measured
[0318] Batch Release Testing of RPE cells produced in Mock runs 4
and 5: To verify that OpRegen.RTM. produced in mock runs 4 and 5,
is comparable to GMP produced OpRegen.RTM., abbreviated
OpRegen.RTM. batch release testing was carried out that included
morphology testing at the end of P2 prior to cryopreservation (IPC
point 10, DS), and viability, total cell number/cryovial, identity
(expression of Bestrophin 1 and MITF), hESC impurity, and
karyotyping at the end of P2 post cryopreservation (IPC point 11,
DP). OpRegen.RTM. produced in Mock runs 4 and 5 passed batch
release criteria. OpRegen.RTM. produced in mock run 2 was not
cryopreserved, and thereby could not be tested.
[0319] Conclusion
[0320] Three mock production runs (mock runs 2, 4, and 5) were
carried out under research grade conditions using the same
GMP-production methods, xeno-free GMP-grade cells (HAD-C 102 hESCs
grown on irradiated CRD008 feeders), xeno-free GMP grade reagents
and GMP grade lab-ware that were used in the GMP production of the
clinical batches. Mock productions 2, 4 and 5 aimed at assessing
the level of hESC impurity along the production and Mock
productions 4 and 5, also aimed at identifying important in process
quality controls.
[0321] Using a qualified TRA-1-60/Oct4 double staining FACS method
(LOD 0.0004%, 1/250,000 and LLOQ of 0.001%, 1/100,000) and a
qualified flow cytometer, hESC impurity in level below assay LOD
was observed at the end of the differentiation phase, in the
negatively selected pigmented cells, three stages prior to the end
of Mock 5 production process. In mock runs 2 and 4, performed prior
to assay qualification using core facility flow cytometer, the
level of hESC impurity was below assay LOD two stages prior to the
end of the production process. In support with this data,
quantitative RT-PCR analysis demonstrated down regulated expression
of the pluripotent hESC genes GDF3 and TDGF to levels similar to
the negative control (OpRegen.RTM. 5C cells) two stages prior to
the end of the production process.
[0322] Identity testing performed three stages prior to the end of
production (isolation of pigmented cells) demonstrated expression
of Bestrophin 1 and MITF by 93% and 96.5% of the immunostained
cells, respectively, as well as expression of CRALBP and ZO-1 (not
quantified). RPE purity testing performed one stage later (i.e. P0,
2 stages prior to the end of the production process), following one
expansion cycle of the negatively selected pigmented cells, showed
that >98.5% of the cells were CRALBP.sub.+PMEL17.sub.+ double
positive by FACS. Similar level of RPE purity (i.e. >99.6%) was
also detected in the drug product. These results were supported by
morphology testing demonstrating typical polygonal shaped
epithelial monolayer morphology and by quantitative RT-PCR analysis
demonstrating upregulated expression of the RPE genes Bestrophin 1,
CRALBP, and RPE65 to levels similar to the positive control
(OpRegen.RTM. 5C cells).
[0323] PEDF, known to be secreted from RPE cells, was measured in
the cell culture medium at various stages along the production
process of mock runs 4 and 5. At the end of the Activin A treatment
(IPC point 4), previously shown by Idelson et al. 2009) to direct
the differentiation towards RPE cells, the level of secreted PEDF
was highly increased (31 fold in mock 4 and 37 fold in mock 5)
relative to the previous production step (induction of spheroid
bodies). PEDF secretion levels continued to increase and peaked at
P0-P1 (1.7-5.8 fold increase relative to the levels after Activin
A). Assessment of the relative area of pigmented cells at the end
of the differentiation process (IPC point 5) was identified as
another important quality control measure for assessment of RPE
differentiation. Using this measure, a 2 fold difference in the
yield of pigmented cells in mock 4 and 5 runs (32.5% in mock 4 and
60% in mock 5) was observed, that was correlated with a similar
difference seen in PEDF secretion at this stage (1,482 ng/ml/day in
mock 4 and 4,746 ng/ml/day in mock 5).
[0324] In conclusion, no TRA-1-60.sub.+Oct4.sub.+hESC impurity
observed as early as 3 stages prior to the end of the production
process. This was correlated with low expression levels of GDF3 and
TDGF, high expression levels of Bestrophin 1, CRALBP and RPE65, and
high levels of Bestrophin 1 and MITF single positive cells, as well
as high CRALBP+PMEL17.sub.+ double positive cells (tested one stage
later). Important safety and efficacy IPCs were identified at
critical production stages.
Example 5
Efficacy Assessment
[0325] Experimental set-up: The present inventors examined whether
subretinal transplantation of the RPE cells generated as described
in Example 4 could delay the progression of RDD in the Royal
College of Surgeons (RCS) rat model.
[0326] 25,000, 100,000 or 200,000 RPE cells were transplanted into
the subretinal space of one eye of RCS rats on post-natal day
(P)21-23 (prior to photoreceptor death onset); BSS+(Alcon) treated
and naive untreated animals served as controls. Groups were
separated into 4 survival ages: post-natal day P60, P100, P150 and
P200. Fundus photography was used to identify bleb formation and
monitor injection quality. Funduscopy was also performed at P60,
P100, P150 and P200. Optomotor tracking was used to measure visual
acuity of all animals at all time points (P60, P100, P150,
P200).
[0327] Focal and full field ERGs were assessed in all study groups
at P60 and P100. At the assigned sacrifice date for each animal,
both eyes were removed, fixed in 4% paraformaldehyde,
cryopreserved, embedded in Optimum Cutting Temperature compound
(OCT) and cryosectioned. Cresyl violet staining was used to
identify and enumerate photoreceptor structural rescue.
Immunofluorescent staining (IF) was used to identify transplanted
cells, assess their fate, their state of proliferation, and their
ability to phagocytose photoreceptor outer segments. In addition
immunofluorescene was used in measurement of host cones rescue.
[0328] The study design is summarized in Table 6 herein below.
TABLE-US-00006 TABLE 6 TIME OF SACRIFICE POST INJECTION Number of
Mice TREATMENT GROUPS (male and female) GRP Total at Study
Initiation # Article # of Cells P60 P100 P150 P200 1 Untreated None
13 11 13 10 2 Vehicle Control None 15 13 16 17 3 RPE Low Dose
25,000 15 15 16 14 4 RPE Medium Dose 100,000 15 15 18 13 5 RPE High
Dose 200,000 15 16 15 13 (MFD)
[0329] Materials and Methods
[0330] Cell counts: Cells were counted before being aliquoted into
appropriate dosage concentrations. Pre-injection cell viability for
all injection time points averaged 94.0%.+-.0.03. Post injection
cell viability averaged 92.4%.+-.0.02.
[0331] Surgery: A small incision was made through the conjunctiva
and sclera using incrementally smaller gauge needles: 18, 22, 25,
and 30. A lateral margin puncture of the cornea was used to reduce
intraocular pressure, to reduced egress of the injected cells. The
glass pipette was then inserted into the subretinal space and 2
.mu.l of suspension injected. The sclerotomy was then sutured
closed. Successful injection of the cells or buffer alone (BSS+)
was confirmed first by manual visualization of a subretinal bleb,
which was subsequently photographed through the use of a fundus
camera (Micron III).
[0332] Optokinetic tracking thresholds: Optokinetic tracking
thresholds were measured and recorded in a blinded fashion.
Repeated measures ANOVA or one-way ANOVA with Fisher's LSD post hoc
analysis was used to analyze OKT data.
[0333] Electroretinagram (ERG): Two forms of ERGs were measured: an
exploratory form of focal ERG where a small spot of light is used
to stimulate a localized area of retina, and a standard style of
full field ERG where the entire visual field is stimulated.
[0334] Histology and Immunohistochemistry: Both eyes from each
animal were harvested, fixed, cryoprotected, embedded, and frozen.
Frozen blocks were cryosectioned at 12 .mu.m. Approximately 60
slides containing 4 sections per slide were obtained.
[0335] Cresyl Violet: Cresyl violet stained sections were examined
for: 1) injection site and suture, 2) evidence of photoreceptor
rescue, 3) evidence of transplanted cells, 4) untoward pathology.
For each slide, maximum outer nuclear layer thickness was also
recorded for quantification of rescue.
[0336] Immunofluorescence (IF): RPE cell treated eye slides
selected for IF were chosen from cresyl violet stained sections
that contained cells in the subretinal space consistent with the
size and morphology of the transplanted human cells. In addition,
protection of the host ONL was used as a secondary criterion. All
IF staining was performed as dual stains with DAPI serving as a
background nuclear stain. At least one slide from every cell
treated animal was used for each run.
[0337] Run #1 was performed using rabbit monoclonal Anti-Melanoma
gp100 (PMEL17, Clone EPR4864; human specific, Abcam cat #ab137062)
co-stained with mouse monoclonal Anti-Nuclei Marker (HuNu, Clone
3E1.3, Millipore, cat #MAB4383) for detecting human RPE and non-RPE
cells.
[0338] Run #2 was performed using rabbit monoclonal Anti-Ki67
(Ki67; Clone EPR3610, human specific, Abcam, cat #ab92742) and
Anti-Nuclei Marker for detecting human proliferating cells.
[0339] Run #3 was performed using rabbit polyclonal Anti-rat Cone
Arrestin (Millipore cat #ab15282) to evaluate sections for cone
counting (see Section 6.8.3). In addition, selected slides were
stained using mouse monoclonal Anti Rhodopsin (Clone Rho 1D4,
Millipore, MAB5356) in combination with PMEL17 to identify
transplanted human cells containing host rhodopsin/outersegments as
a measure of their phagocytic activity.
[0340] Cone Counting: Confocal z-stack images were acquired from
sections of retina obtained from all cell transplanted eyes and
from age-matched saline injected controls. Sections from cell
injected eyes were chosen in the area of photoreceptor rescue as
defined using the previously evaluated cresyl violet stained
sections. Cones were counted by 3 observers in a blinded fashion.
The three counts were then averaged and counts compared between
dosage groups and age.
[0341] Rhodopsin ingestion: A potential mechanism of rescue
employed by the transplanted cells is to ingest photoreceptor outer
segments and shed debris. Removal of the debris zone reduces the
toxic stress on the photoreceptors and thus, aids in sustaining
photoreceptor survival. Here, the present inventors selected
specific animals for evaluation of rhodopsin ingestion by the RPE
cells based on the cell survival and photoreceptor protection
indices. This evaluation was performed using
immunofluorescence.
[0342] Results
[0343] Fundus Imaging: Fundus images collected at necropsy of cell
treated eyes revealed hyper and hypo-pigmented areas of the retina
that corresponded to the location where subretinal blebs were
formed during surgery; the location at which cells were deposited
in the subretinal space (FIGS. 19A-C). These patchy areas were not
evident in BSS+ injected or non-injected eyes.
[0344] Optokinetic tracking thresholds: OKT thresholds were rescued
in all cell treated groups at all ages (FIG. 20). Cell-treated
groups outperformed un-operated or saline injected eyes at all
ages. There was a significant dose dependent effect between the low
dose (25K) and the two larger doses (100K (p<0.0001) and 200K
(p<0.0001)), especially at the later ages, but no clear benefit
to the OKT from the high dose (200K) over the intermediate (100K)
dose was observed (p=0.5646). While OKT thresholds were rescued in
all cell treated groups, the absolute visual acuity values slowly
declined with time. Untreated and saline injected animals' OKT
thresholds continue to decline over the course of the study. BSS+
injected eyes were not different from naive untreated group
(p=0.6068) and untreated fellow eyes.
[0345] Focal ERG: Focal ERG's were measured in all (n=252)
experimental rats at .about.P60. Individual animals treated with
RPE cells performed well and significantly outperformed controls,
as illustrated in FIG. 21A.
[0346] Fullfield ERG: Full field ERG's were measured from 125 RCS
rats at P60 and from 63 RCS rats at P100. Individual animals
treated with RPE cells performed well and significantly
outperformed controls, as illustrated in FIG. 21B.
[0347] Cresyl Violet staining: An exemplary photomontage of a
cresyl violet stained section is presented in FIG. 22A.
Representative images from BSS+ injected and cell treated (images
from multiple groups) eyes are presented in FIG. 22B.
[0348] Outer nuclear layer thickness (ONL) was measured as the
primary indicator of photoreceptor rescue. Data was recorded as
maximum number of photoreceptor nuclei present in each dose group
across ages (FIG. 23). Cell treated groups had significantly higher
ONL thickness at P60, P100 and P150 (All p<0.0001) than BSS+
treated eyes. In terms of percentage of animals with evidence of
photoreceptor rescue, 76-92% of animals at P60, 80-90% at P100,
72-86% at P150, and 0-18% at P200 had evidence of
photoreceptor.
[0349] Immunofluorescence: Transplanted RPE cells were positively
identified by immunofluorescence in animals of each survival age
(FIG. 24), however, the number of animals with identified cells
decreased as age increased. Repeat staining of additional slides in
animals that did not originally reveal transplanted cells resulted
in additional animals identified with positive cells, but not in
all cases.
[0350] Despite not finding transplanted cells in all animals by IF
analysis, ONL thickness measurement results indicated 70-90% of
cell treated animals had significant photoreceptor rescue,
confirmed with OKT rescue, suggesting that most treated eyes
contained transplanted cells at some point. The proliferation
marker Ki67 was used to identify proliferating human cells. Ki67
positive human cells were not observed (FIG. 24).
[0351] Cone Counting: Cone counts in animals that received cell
transplants were significantly better than control eyes (FIG. 25;
p=<0.0001 for each comparison). In general, there was no
difference between cone counts across the low, middle and high
dosage of cells. A representative image from each age is presented
in FIG. 24.
[0352] Rhodopsin ingestion: In each case tested (n=6),
fluorescently labeled rhodopsin was observed within the
transplanted RPE cells (FIGS. 26A-J). This confirms the
transplanted cells do ingest outer segment debris post
transplantation.
[0353] Conclusion
[0354] When transplanted into the subretinal space of RCS rats, RPE
cells rescued visual acuity in the RCS rat over that of controls at
all ages tested. ERG responses were protected when the graft was
large enough or in an area of retina accessible for assessment. Rod
and cone photoreceptors were rescued in the area of the grafts for
up to 180 days post-transplantation. Collectively, this data
demonstrates that OpRegen.RTM. maintain the functional and
structural integrity of the host retina for extended periods. Thus,
OpRegen.RTM. hold significant potential for the treatment of human
RPE cell disorders such as RP and AMD.
Example 6
Stability of RPE Cells
[0355] Short-Term Stability
[0356] Formulated RPE cells (generated as described in Example 4)
in BSS plus were prepared at a final volume of 600-1000 .mu.l per
vial. Short term stability was tested at time points 0, 4, 8 and 24
hours. Cells were found stable at all time points.
[0357] RPE cell viability and cell concentration were stable at the
8 hour incubation time point for all dose formulations; percent
average viability (.+-.SD) for the following concentrations: [0358]
Low concentration (70.times.10.sup.3 per 100 .mu.l BSS plus)
changed from 93%.+-.5 at time point 0 hours to 91%.+-.1 at time
point 8 hours, a non-significant decrease. [0359] High
concentration (70.times.10.sup.3 per 100 .mu.l BSS plus) changed
from 92%.+-.3 at time point 0 hours to 91%.+-.2 at time point 8
hours, a non-significant decrease.
[0360] For the medium concentration (250.times.10.sup.3 per 100
.mu.l BSS plus) that was tested there was no significant change
throughout the time points.
[0361] The overall range for all time points and formulated doses
was between 88%-97% from time point 0 hours to 8 hours, when
averaging all results for time point 0 hours (93%.+-.3) and time
point 8 hours (91%.+-.1) a decrease of 2% was found.
[0362] No significant changes in the cell concentration were
observed, in either time points or formulated doses. Cell
concentration did not change in all 3 studies other than a small
decrease seen in one batch in the high dose (2%).
[0363] Appearance of the different dose formulations did not change
throughout the tested time points; cell suspension was free of
foreign particles and non-dissociated aggregates.
[0364] Identity and purity of each formulated RPE cell dose at all
tested time points were stable up to 24 hours and were within the
batch release criteria. At 8 hours (for all formulated RPE cell
doses), the level of MITF and Bestrophin positive cells was in the
range of 86-97% and 90-94%, respectively, and the level of
CRALBP+PMEL17+ double positive cells was in the range of
98.35-99.64%.
[0365] Formulated RPE cell doses maintained their potency in all
tested time points (4, 8, 24 hours), both secreting high levels of
PEDF and forming a polarized RPE monolayer with a polarized
secretion of PEDF predominantly to the apical side and VEGF to the
basal side. Results for the tested time points 8 hours: TEER was in
the range of 376-724 ohms, PEDF apical to basal ratio in the range
of 2.77-5.70 and VEGF basal to apical ratio in the range of
2.04-3.88.
[0366] Sterility was kept at all incubation time points for all
cell dose formulations.
[0367] These results support OpRegen.RTM. cell stability in final
formulation at all clinical doses for at least 8 hours when kept at
2-8.degree. C. A safety margin of up to 24 hours exists based on
partial data collected (identity, sterility, and medium dose
potency).
[0368] Results of the short term stability assay are summarized in
Table 7 below.
TABLE-US-00007 TABLE 7 LOW DOSE MID DOSE HIGH DOSE ACCEPTANCE
70,000 250,000 700,000 TEST CRITERIA cells/100 ml cells/100 ml
cells/100 ml Cell Viability .gtoreq.70% 91 .+-. 1 (n = 3) 92 (n =
1) 91 .+-. 1.5 (n = 3) Cell Dose .+-.40% from 91.3 .+-. 30 (n = 3)
103 (n = 1) 104 .+-. 5.7 (n = 3) initial dose MITF Positive Cells
.gtoreq.80% 90 (n = 2) 93 (n = 1) 96 (n = 2) Bestrophin 1 Positive
Cells .gtoreq.80% 94 (n = 2) 92 (n = 1) 92 (n = 2)
CRALBP.sup.+PMEL17.sup.+ Cells .gtoreq.95% 99.3 .+-. 0.15 (n = 3)
99.5 (n = 1) 99 .+-. 0.65 (n = 3) Barrier Function, TER (.OMEGA.)
For 605 (n = 2) 724 (n = 1) 410 (n = 2) Polarized PEDF Secretion
Information 3.4 (n = 2) 3.5 (n = 1) 4.5 (n = 2) (Apical/Basal) Only
Polarized VEGF Secretion 3.3 (n = 2) 2.2 (n = 1) 2.3 (n = 2)
(Basal/Apical) Sterility USP<71> Negative Negative Negative
Negative Appearance No foreign Pass Pass Pass particles and/or
non-dissociated aggregates
[0369] Long-term stability: Three batches of RPE cells were frozen
in vapor phase liquid nitrogen. Testing of the long-term stability
in cryopreservation started after the freezing date. Results
provided are following three years of freezing. The following
parameters are being tested: viability, cell number, RPE identity
(% Bestrophin 1 and % MITF positive cells), RPE purity (FACS %
CRALBP+PMEL17.sub.+RPE cells), potency (polarization and PEDF
secretion), karyotype analysis and sterility. At each time point,
the required number of vials are thawed and the cells are prepared
for the assays as described herein.
[0370] Results of the long term stability assay are summarized in
Table 8 below.
TABLE-US-00008 TABLE 8 TEST 0-3 Months 19-21 Months 34-36 Months
Cell Viability 86 .+-. 2 (n = 3) 87 .+-. 4 (n = 5) 89 .+-. 2 (n =
6) Total Cells/Vial 1.44 .+-. 0.13 (n = 3) 1.13 .+-. 0.2 (n = 5)
1.13 .+-. 0.2 (n = 6) Identity: MITF Positive 84 95 86 (n = 2)
Cells Bestrophin 1 91 90 93 (n = 2) Positive Cells Purity:
CRALBP.sup.+PMEL17.sup.+ 99.8 NA 99.4 Cells Potency: Barrier
Function, 616 368 396 .+-. 200 (n = 3) TER (.OMEGA.) Polarized PEDF
Secretion 3.93 3.86 3.05 .+-. 0.04 (n = 3) (Apical/Basal) Polarized
VEGF Secretion 2.74 1.86 2.90 .+-. 0.50 (n = 3) (Basal/Apical)
Safety: Karyotyping Normal Normal NA Sterility USP<71>
Negative NA NA
[0371] Results
[0372] Viability, total cell number/vial and RPE identity were
maintained throughout the three year period. In addition, as
indicated, data demonstrated potency and purity at levels similar
to the ones collected prior to preservation.
[0373] A normal karyotype was observed 4 years post
cryopreservation. This indicates that long-term storage in vapor
phase thus far did not have any deleterious effects on RPE genomic
stability.
[0374] Sample sterility was demonstrated by testing for the absence
of bacterial/fungal growth in all clinical batches at 3 months.
Another batch was tested negative 4 years post cryopreservation.
Based on these uniformly acceptable stability results, covering a
period of three years of stability testing thus far, it is
concluded that the RPE cellular product is stable for at least
three years when stored at a temperature <-180.degree. C. in the
vapor phase of liquid nitrogen.
Example 7
Safety and Biodistribution
[0375] The objectives of the study were to evaluate survival,
biodistribution, and safety of RPE cells (generated as described in
Example 4) following subretinal administration in male and female
NOD-SCID mice over a 6-month study duration.
[0376] NOD-SCID mice (NOD.CB17-Prkdcscid), 5-6 weeks of age at the
time of injection, were injected with either BSS Plus (Vehicle
Control) or with two doses of RPE cells: 50.times.10.sup.3 cells or
100.times.10.sup.3 cells (maximal feasible dose), suspended in 1
.mu.L BSS Plus. RPE was administered into the subretina via the
transvitreal route (the proposed clinical route of administration)
using a 33G Hamilton needle. A single dose of 50.times.10.sup.3
cells or 100.times.10.sup.3 cells was injected to one eye, while
the fellow eye served as an internal control. Each dosing session
contained mice (males and females) from each group. Mice included
in the study after pretest, were randomly assigned to the various
test groups. Two randomizations were performed. A measured value
randomization procedure, by weight, was used for placement into
treatment groups prior to vehicle/test article administration.
Following administration, animals suitable for use on study were
transferred to the target study using a sequential randomization
for placement into the final treatment groups. Mice with ocular
abnormalities, abnormal clinical observations or weighing less than
16 gram at pretest and mice undergoing non-successful subretinal
RPE injection were excluded from the study.
[0377] Study Measurements: Assessment of RPE safety in this study
was based on animal mortality, clinical observations, body weight,
ophthalmologic examinations, clinical pathology (hematology and
blood chemistry), gross pathological macroscopic evaluations, organ
weights (absolute and relative to body and brain weights),
histopathological evaluation of eyes and various organs. Assessment
of survival and biodistribution of RPE was performed by
histopathological and fluorescence immunostaining evaluations of
eyes and various organs and qPCR analysis. The following
measurements were performed: [0378] Clinical observation; [0379]
Body weight; [0380] Ophthalmologic examinations (including
macroscopic and biomicroscopic examinations); [0381] Surgical
microscopic examination of subretinal injection quality using the
LEICA M80 Stereo microscope (funduscopy); [0382] Complete blood
count and blood chemistry; [0383] Necropsy and gross pathology;
[0384] Organ weight (absolute and relative to body and brain
weights); [0385] Collection, fixation, and paraffin blocking of
treated and non-treated contralateral eyes including optic nerve;
[0386] Blinded H&E histopathology of eyes and tissues (sternum
bone with bone marrow, brain, heart, kidneys, liver, lung,
mandibular lymph nodes, spinal cord, spleen, thymus, masses and
gross lesions); [0387] Blinded semi quantitation of pigmented cells
in H&E stained slides; [0388] Blinded immunostaining of
selected slides adjacent to a representative H&E slide
demonstrating pigmented cell graft in the eye for a human marker
(human nuclei) plus an RPE marker (human PMEL17) and assessment of
human RPE and non-RPE cells, human marker (human nuclei) plus a
proliferation marker (human Ki67) and assessment of human and
non-human proliferating cells, and RPE marker (RPE65) plus
proliferation marker (human Ki67) and assessment of RPE and non-RPE
human proliferating cells; [0389] Blinded immunostaining of
selected slides adjacent to a representative H&E slide
demonstrating teratoma, tumor, abnormal cells and lesions for a
human marker (human nuclei) to exclude human origin; [0390]
Collection and extraction of genomic DNA from blood, bone marrow
(collected from femurs), brain, left and right eyes with optic
nerves, heart, left and right kidneys, liver, lung, mandibular
lymph nodes, ovaries, skeletal biceps femoris muscle, spinal cord,
spleen, testes, and thymus and qPCR analysis of human beta globin;
[0391] H&E histopathology on tissues (other than the above)
found positive for human beta globin in animals from the same group
and time point.
[0392] Results
[0393] There were no RPE-related toxicologic findings in the
in-life examinations which included detailed clinical observation,
body weight, ophthalmologic examination and clinical pathology
comprised of hematology and serum clinical chemistry. The
observation of "Eye discolored, dark" in the left eye with an
albino background was found in mice treated with pigmented RPE
cells at both dose levels in the detailed clinical observation and
ophthalmologic examination. Ophthalmologic examination of the
surviving animals indicated that this observation consisted of
mid-vitreal, darkly pigmented foci. The pigmented foci were
distributed randomly along a line extending from the temporal
posterior lens capsule to the nasal retinal surface. These foci
were interpreted to be RPE cells escaping from the injection
cannula upon its removal from the eye following injection, as
supported by the vitreal reflux seen during injection or RPE cells
leaking into the vitreous humor subsequent to subretinal
implantation.
[0394] All of the ocular lesions observed on this study were
considered to arise secondary to anesthesia, the surgical injection
procedure, or incidentally as age-related changes. The finding of
multiple pigmented foci within the vitreous humor suggests that RPE
cells may be viable within the vitreous body. The presence of
pigmented cells in the vitreous body in some of the RPE-treated
animals was confirmed at the microscopic level.
[0395] In terms of biodistribution as evaluated by qPCR using a set
of human beta-globin gene probe/primers, at the 2-week, 2-month,
and 6-month intervals, the left eyes treated with
100.times.10.sup.3 OpRegen.RTM. cells were positive for RPE DNA in
8/12, 11/12, and 16/16 animals with group mean levels at 38, 47 and
249 copies/.mu.g total eye DNA, respectively, indicating a trend of
increase over time. There was no significant difference between
males and females. In these animals, RPE DNA was not detected in
the untreated right eyes and all the non-eye tissues, which
included blood, femoral bone marrow, brain, heart, kidneys, liver,
lung, mandibular lymph nodes, ovaries, skeletal biceps femoris
muscle, spinal cord, spleen, testes, and thymus, except for the
spinal cord (27 copies/.mu.g DNA) from one 2-week male animal and
the skeletal muscle (16 copies/.mu.g DNA) and spinal cord (below
level of qualification) from one 2-week female animal (probably due
to inadvertent contamination by exogenous human DNA during DNA
extraction from these tissues).
[0396] RPE-related macroscopic changes were limited to black
discoloration or black foci in the left eye of a few animals at the
2 and 6-month intervals, consistent with in-life clinical
observation and/or ophthalmologic examination. These changes
correlated to pigmented cells and were not considered adverse as
determined by microscopic examination of surviving animals in the
high-dose group and of the animals euthanized in extremis and found
dead in both dose groups. Pigmented cells were present in the
treated left eye in nearly all of the surviving mice examined at
each time point in the high dose group (at the subretinal space in
11/12, 12/12 and 16/16 in the 2-week, 2-month, and 6-month
intervals), as well as the animals euthanized in extremis or found
dead in both low and high dose groups. The most common locations of
the pigmented cells were the subretinal space and the vitreous body
as confirmed by immunostaining of human cell- and RPE-specific
biomarkers. In the subretinal space, pigmented cells tended to be
restricted to the injection site at the earlier time points,
whereas at the later time points they were present at locations
distant from the injection sites, suggesting local cell spreading.
There was a slight increase in average total number of pigmented
cells per eye at the 6-month time point compared to 2-week or
2-month time points in males. This increased number of pigmented
cells of human origin was supported by the qPCR analysis.
[0397] Long-term engraftment of the RPE cells is illustrated in
FIG. 27A. Pigmented cells stain positive for Human Nuclei and
PMEL17 in NOD-SCID subretinal space 9 months post transplant.
[0398] FIG. 27B is a photograph illustrating the clustered at the
place bleb following injection. FIG. 27C is a photograph
illustrating the subsequent spreading of the cells into a monolayer
following injection.
[0399] RPE was not associated with any organ weight changes. There
were no macroscopic and microscopic changes in the untreated right
eyes and the non-eye organs examined in this study which included
brain, heart, kidneys, liver, lung, mandibular lymph nodes, spinal
cord, spleen, and thymus. Anti-human nuclei biomarker antibody
stain (Human Nuclei) was observed in 64%, 36%, and 73% of the
tested left eyes at 2-week, 2-month, and 6-month time points,
respectively, in the animals examined in the high dose group.
[0400] The highest detection level for Human Nuclei was noted in
pigmented cell populations within the subretinal space followed by
the vitreous body. Anti-human RPE-specific biomarker PMEL17
staining was observed in most of the animals tested whereas another
RPE-specific biomarker, RPE65, had various levels of detection at
the different time points. These RPE-specific biomarkers were
mostly detected in the subretinal space and less in the vitreous
body. Human cell proliferation biomarker Ki67 was detected in only
a few cells in a small number of animals, mainly in pigmented cells
within the vitreous body and less within the subretinal space. The
incidence of Ki67 positivity decreased over time with only one
animal at 6 month. The Ki67-positive cells were not associated with
any abnormal morphology.
[0401] Several microscopic changes were noted at the injection site
across all the time points and all the study groups and considered
related to the surgical injection procedure. Some of these changes
were slightly more prominent in animals examined in the high dose
group at 6 months. For example, retinal detachment was noted in one
animal and the incidence or severity of retinal
degeneration/atrophy or fibroplasia was slightly increased compared
to the vehicle control group.
[0402] There were no RPE-dependent effects on animal mortality rate
and survival.
[0403] Conclusion
[0404] No local or systemic toxicologic, lethal, or tumorigenic
effects were observed in the NOD/SCID animal model during the
6-month study period following single injection of RPE at dose
levels of up to 100,000 cells/.mu.l/eye. Biodistribution of RPE
cells was restricted to the treated left eye with local subretinal
cell spreading from the subretinal injection site as a function of
time. RPE cells were present predominantly in the subretinal space
followed by the vitreous body in most of the animals examined in
the high dose group at 2-week, 2-month, and 6-month intervals, with
variable positivity in immunostaining by antibodies against the
human nuclei and/or human RPE-specific biomarkers. The persistence
of RPE cells in the eye was estimated to be at least 6 months with
very limited cell proliferation. The limited proliferation took
place mostly in the vitreous body and had no adverse effects. There
was evidence that the number of RPE cells increased in the treated
eye over time, although this was accompanied by decreased
proliferation incidence in the subretinal population examined.
Expression of both RPE specific markers RPE65 and PMEL17 was
predominantly in RPE cells within the subretinal space as opposed
to those within the vitreous body, where most of Ki67-positive cell
incidences were found. The latter suggests that the increase in RPE
cells over time is limited to the vitreous space and that the
expression of specific RPE65 and PMEL17 RPE markers may be
regulated by the microenvironment. In conclusion, based on the data
presented above, there are no serious safety concerns related to
the injection of the presently described RPE cells as compared to
vehicle control group.
Example 8
Expression of Pax-6 in the RPE Cells
[0405] Objective: Development of a FACS based method for assessing
the level of PAX-6 in human retinal pigment epithelial (RPE)
cells.
[0406] Materials and Methods
[0407] Frozen RPE cells (generated as described in Example 4, were
thawed spun down, re-suspended in 1 ml PBS minus, filtered through
a 35 .mu.M cell strainer and counted with the NC-200 cell counter.
The cell concentration was adjusted to .about.1.times.10.sup.6
cells/ml in PBS minus. 1 .mu.l/ml FVS450 was added to each ml cell
suspension followed by vortexing and incubation for 6 minutes at
37.degree. C. FVS450 was quenched with 0.1% BSA(-Ig)-PBS minus, and
re-suspended in 0.1% BSA(-Ig)-Fc-block (5 min at RT) to block all
Fc-epitopes on the cells. Cells were then fixed and stained with
anti-Pax-6 antibody (AF647 Cat #562249).
[0408] Results
[0409] As can be seen in FIG. 29, cells at P0 and P2 are positive
for PAX6 (81.5%-82.5% at P0 and 91.3%-96.1% at P2). P2 is the
passage at the end of the production process and P0 is two
expansion stages earlier. The data was shown to be consistent
across batches, as shown in FIGS. 29 and 30. In addition, the
present inventors showed by FACS analysis that the RPE cells double
stained for PAX-6 and CRALBP (FIG. 31).
Example 9
Identification of Proteins Secreted by the RPE Cells
[0410] Objective: To identify a signature of proteins (known and
new) secreted by the OpRegen.RTM. (RPE cells) that can be used as a
batch release potency assay as well as a process control assay.
[0411] Supernatants were collected from RPE cells (generated as
described in Example 3) that were cultured under different culture
conditions indicated below. Supernatants were then screened using
the G6 and G7 RayBiotech arrays according to manufacturer's
instructions after an overnight incubation of the supernatants with
the related array.
[0412] 1. RPE drug product cells post thawing cultured for 4 and 14
days on 12-well plate (0.5.times.10.sup.6 cells/well at Passage 3)
(referred to herein as OpRegen.RTM.).
[0413] 2. RPE drug product cells post thawing cultured for 14 days
on 12-well plate and then cultured for 3 weeks on a Transwell (as
per AM-RPE-15) and demonstrated TEER >500.OMEGA.. Supernatants
were taken from the apical and basal chambers.
[0414] 3. Cells generated according to the protocol described in
Example 3, prior (QC3) and post (QC4) Activin A treatment.
[0415] 4. Nutristem medium (Nut-) without addition of TGF.beta. and
FGF.
[0416] Supernatants were also collected from the following cell
cultures and tested by ELISA:
[0417] 1. OpRegen.RTM. drug product cells post thawing that were
each cultured for 14 days on 12-well plate and then cultured for 3
weeks on a Transwell (as per AM-RPE-15) and demonstrated TEER of
355.OMEGA. and 505.OMEGA., respectively. Supernatants were taken
from day 14 (passage 3) and from the apical and basal chambers.
[0418] 2. RPE 7 cells post thawing that were cultured for 14 days
on 12-well plate (0.5.times.10.sup.6 cells/well at Passage 3).
[0419] 3. Mock VI cells at the end of Passage 1 of the production
process that were grown on laminin521 following Enzymatic or
Mechanical isolation (as described in Example 3). These cells were
tested for potency as per AM-RPE-15 and supernatants were collected
from cells at Day 14 on 12 well plate (passage 2) and cells after 3
weeks on transwell from the apical and basal chambers.
[0420] 4. Fetal HuRPE cells at Passage 3 Days 4 and 14
(0.5.times.10.sup.6 cells/well).
[0421] ELISA test validation was performed according to
manufacturer's instructions related to each ELISA kit. In each
protocol, incubation with the supernatants was overnight.
[0422] Study design: Supernatants were collected from the cells
that were cultured under different culture conditions and kept at
-80.degree. C. Following protein array analysis, validation of the
hits was measured by ELISA.
[0423] Results
[0424] The G7 array results are provided in Table 9 herein
below.
TABLE-US-00009 TABLE 9 G7 Nut (-) Day 4 Day14 TW Apical TW Basal
QC3 QC4 POS 18,132 18,132 18,132 18,132 18,132 18,132 18,132 NEG 69
65 15 41 79 23 45 Acrp30 18 4,739 46 22 114 102 4 AgRP 56 61 62 72
75 57 94 Angiopoictin-2 15 35 13 22 32 373 306 Amphiregulin 28 24
32 36 30 27 32 Axl 15 30 100 365 29 41 103 bFGF 15 22 23 95 20 211
28 b-NGF 11 29 31 24 31 61 30 BTC 41 58 46 47 54 127 59 CCL-28 37
42 40 36 34 88 60 CTACK 57 58 80 71 79 68 73 Dtk 16 17 17 21 21 23
24 EGF-R 11 61 174 227 156 138 77 ENA-78 23 34 27 31 34 36 36
Fas/TNFRSF6 19 22 25 24 33 21 23 FGF-4 16 19 19 20 25 14 22 FGF-9
19 17 27 21 27 21 26 GCSF 200 246 235 233 246 245 262 GITR-Ligand
47 54 52 50 53 46 56 GITR 24 26 26 29 29 28 24 GRO 121 367 224 952
400 549 472 CRO-alpha 65 61 79 64 77 65 85 HCC-4 50 72 40 38 43 40
85 HGF 19 20 20 31 18 239 35 ICAM-1 13 20 24 27 17 106 56 ICAM-3 9
14 14 8 12 2 9 IGFBP-3 18 22 25 84 24 25 601 IGFBP-6 13 172 39 167
59 107 66 IGF-I SR 27 26 27 27 29 23 33 IL-1 R4/ST2 43 36 44 41 45
34 111 IL-1 RI 61 56 50 54 59 48 65 IL-11 54 58 51 60 89 55 64
IL-12 p40 10 16 13 12 17 18 12 IL-12 p70 15 18 27 19 18 18 20 IL-17
47 57 67 51 52 50 55 IL-2 Rapha 57 67 115 62 66 64 69 IL-6 R 12 25
42 15 15 81 18 IL-8 107 119 113 237 135 993 226 I-TAC 14 20 23 18
25 26 24 Lymphotactin 20 26 27 23 24 19 23 MIF 27 261 2,712 3,463
515 4,300 3,736 MIP-1alpha 26 24 25 29 27 23 25 MIP-1beta 18 22 20
17 23 28 1,056 MIP-3beta 19 21 17 19 23 15 17 MSP-alpha 21 34 26 25
25 37 33 NT-4 10 14 11 12 13 9 15 Osteoprotegerin 16 48 4,622 191
33 830 593 Oncostatin M 40 46 44 52 61 53 39 PIGF 46 111 110 89 75
284 336 sgp130 16 93 199 393 40 222 564 sTNF RII 13 15 12 13 18 40
10 sTNF-RI 123 449 675 1,703 163 293 203 TECK 50 61 60 52 54 75 59
TIMP-1 130 1,223 1,909 1,674 1,948 2,006 1,798 TIMP-2 15 571 621
1,937 753 483 776 Thrombopoietin 48 48 47 47 48 54 39 TRAIL R3 39
100 100 310 56 572 314 TRAIL R4 23 22 21 18 21 46 20 uPAR 68 161 67
148 65 276 87 VEGF 14 508 689 559 554 546 592 VEGF-D 20 21 23 20 22
25 19
[0425] The G6 array results are provided in Table 10 herein
below.
TABLE-US-00010 TABLE 10 G6 Nut (-) Day 4 Day 14 TW Apical TW Basal
QC3 QC4 POS 12,843 12,843 12,843 12,843 12,843 12,843 12,843 NEG 18
5 20 8 10 2 12 Angiogenin 4 3,006 3,152 423 1,749 2,838 3,574 BDNF
12 8 12 9 9 8 9 BLC 14 17 18 11 17 10 12 BMP-4 9 38 9 9 6 6 6 BMP-6
6 3 4 2 4 3 1 CK beta 8-1 9 7 8 9 10 6 8 CNTF 79 72 68 68 68 75 78
EGF 5 8 6 8 7 10 1 Eotaxin 9 13 11 11 12 11 12 Eotaxin-2 9 11 8 4 7
7 8 Eotaxin-3 58 53 62 42 59 47 59 FGF-6 7 4 7 1 9 0 7 FGF-7 9 9 16
14 13 9 14 Flt-3 Ligand 49 51 50 46 54 49 46 Fractalkine 6 3 6 4 4
5 5 GCP-2 8 8 9 8 13 16 7 GDNF 10 11 12 12 9 10 11 GM-CSF 63 52 58
50 52 51 60 1-309 5 7 9 6 6 5 7 IFN-gamma 96 77 72 71 89 80 79
IGFBP-1 7 19 21 25 9 7 10 IGFBP-2 10 274 432 490 257 602 442
IGFBP-4 9 11 10 8 7 6 4 IGF-I 9 13 13 14 13 14 16 IL-10 59 59 54 43
57 60 66 IL-13 81 77 66 62 70 69 75 IL-15 56 55 62 46 58 57 55
IL-16 3 3 1 6 3 3 4 IL-1alpha 77 76 63 72 78 77 71 IL-1beta 8 12 16
12 8 8 14 IL-1ra 65 58 68 58 60 55 59 IL-2 54 53 62 51 54 51 190
IL-3 56 49 52 50 52 51 177 IL-4 7 6 7 7 6 6 10 IL-5 81 79 82 67 87
76 80 IL-6 309 429 280 1,053 386 2,704 377 IL-7 64 56 62 59 63 57
63 Leptin 15 19 14 17 15 23 17 LIGHT 8 12 10 5 11 7 8 MCP-1 67
3,046 1,460 4,269 3,963 5,061 2,876 MCP-2 16 19 22 22 22 21 21
MCP-3 8 10 10 9 8 62 8 MCP-4 9 11 10 7 8 11 7 M-CSF 19 18 13 14 17
21 19 MDC 9 8 8 7 7 8 7 MIG 34 28 31 29 31 29 52 MIP-1-delta 8 8 8
6 6 6 0 MIP-3-alpha 8 8 8 7 7 33 72 NAP-2 7 11 12 8 7 6 10 NT-3 12
11 10 12 12 11 9 PARC 60 60 56 53 60 57 57 PDGF-BB 13 17 20 15 20
23 21 RANTES 6 63 15 8 13 35 11 SCF 5 14 4 3 11 17 6 SDF-1 20 25 26
20 22 22 22 TARC 11 14 12 12 12 12 10 TGF-beta 1 82 79 83 81 75 85
77 TGF-beta 3 6 11 5 6 4 8 4 TNF-alpha 86 89 84 78 81 86 81
TNF-beta 82 78 84 80 86 83 77
[0426] RPE secreted proteins can be divided into 3 functional
groups: 1) Angiogenic proteins such as VEGF and Angiogenin, 2)
Extracellular matrix regulators such as TIMP-1 and TIMP-2, and 3)
Immunomodulatory proteins such as IL-6, MIF, sgp130, sTNF-R1,
sTRAIL-R3, MCP-1, and Osteoprotegerin. The receptor tyrosine kinase
Ax1 was also found to be secreted by the RPE cells. 6 proteins that
demonstrated high levels of secretion and/or demonstrated a
polarized secretion (apical/basal) pattern were selected for
validation by ELISA (angiogenin, TIMP-2, MIF, sgp130, sTNF-R1 and
sTRAIL-R3). The array data also demonstrated secretion of VEGF as
seen in the polarization assay.
[0427] Angiogenin: Protein array data demonstrated increased
secretion of angiogenin along the production process (Tables 9 and
10). These results were confirmed by ELISA demonstrating that the
level of angiogenin secreted by differentiating cells that were
treated with nicotinamide prior to the addition of Activin A was
0.52 ng/mL, whereas after the 2 weeks treatment with nicotinamide
and Activin A, angiogenin secretion level increased to 0.91 ng/mL
(FIG. 32A). RPE cells which were cultured for 2 weeks in a 12 well
plate (0.5.times.10.sup.6 cells/well; Passage 3) post thawing
secreted angiogenin (FIG. 32B). Polarized RPE cells (week 3 on
transwell; TEER >350.OMEGA., PEDF apical/basal and VEGF
basal/apical ratios >1) secreted angiogenin in a polarized
manner to the basal side with low to no secretion to the apical
side (basal angiogenin levels were in the range of 0.1-0.25 ng/mL
and apical angiogenin levels in the range of 0.05-0.12 ng/mL; FIG.
32B). RPE 7 cells generated according to Idelson et al., 2009 were
unable to generate barrier function in the transwell system (TEER
below 100.OMEGA.) although could secrete VEGF and PEDF. The ability
of RPE7 cells to secrete angiogenin was tested when plated in a 12
well plate for 14 days. RPE7 secreted angiogenin on day 14 of
culture in a level that is within the range of the RPE cells
generated as described herein (FIG. 32C).
[0428] TIMP-1 and TIMP-2 Secretion: Protein array screen
demonstrated secretion of TIMP-1 and TIMP-2 from polarized and
non-polarized RPE cells (FIG. 33A-E). Interestingly, the array data
showed polarized secretion of TIMP-2 to the apical side and TIMP-1
to the basal side (FIG. 33A). ELISA data confirmed that TIMP-2 is
secreted mainly to the apical side by all RPE batches tested so far
(FIGS. 33C-D apical range of 69.9-113.3 ng/mL and basal range of
11.9-43.7 ng/mL). TIMP-2 was also secreted by non-polarized
OpRegen.RTM. cells in levels similar to the levels secreted by
normal human fetal RPE cells (HuRPE, ScienCell) (FIGS. 33C-E). RPE
7 cells also secreted TIMP-2 in levels similar to the OpRegen.RTM.
cells (FIGS. 33C-E). Interestingly, very low levels of TIMP-2 were
detected along the production process at QC3 and QC4 checkpoints
(FIG. 33B).
[0429] Sgp130 Secretion by OpRegen.RTM. Cells: Protein array data
demonstrated increased secretion of sgp130 along OpRegen.RTM.
production process as seen in the IPC/QC check points 3 and 4
(Tables 9 and 10). ELISA data confirmed higher levels of sgp130
secretion following 2 weeks Activin A treatment (IPC/QC4; 1.64
ng/mL) as compared to the levels secreted by the cells following
nicotinamide treatment prior to the addition of Activin A (IPC/QC3;
0.68 ng/mL) (FIG. 34A). OpRegen.RTM. cells which were cultured for
2 weeks in a 12 well plate (0.5.times.10.sup.6 cells/well; Passage
3) post thawing secreted sgp130 (FIGS. 34B-C). RPE 7 cells cultured
under similar conditions secreted sgp130 in levels that were within
the range of OpRegen.RTM. cells (1.0 ng/mL at day 14; FIG. 34D).
Fetal HuRPE cells secreted low sgp130 levels both on day 4 and on
day 14.
[0430] Polarized OpRegen.RTM. cells secreted sgp130 in a polarized
manner to the apical side with low to no secretion to the basal
side (apical sgp130 secretion levels were between 0.93-2.06 ng/mL
and basal sgp130 levels were in the range of 0-0.2 ng/mL; FIGS.
34B-C).
[0431] Shed sTNF-R1: Very low levels of shed sTNF-R1 were detected
by ELISA in the supernatant of differentiating cells prior (IPC/QC3
0.01 ng/mL) and post two weeks treatment with nicotinamide and
Activin A (IPC/QC4 0.02 ng/mL) (FIG. 35A). OpRegen.RTM. cells which
were cultured for 2 weeks in a 12 well plate (0.5.times.10.sup.6
cells/well; Passage 3) post thawing contained sTNF-R1 in the
supernatant of culture day 14 (FIGS. 35B-C). HuRPE cells cultured
under similar conditions had similar levels of sTNF-R1 in their
culture supernatant while RPE 7 cells demonstrated relatively low
sTNF-R1 levels (FIG. 35D).
[0432] Polarized OpRegen.RTM. cells secreted shed sTNF-R1 in higher
levels to the apical side (apical and basal sTNF-R1 levels were in
the range of 0.22-1.83 ng/mL and 0.01-0.11 ng/mL, respectively;
FIGS. 35C-D).
[0433] sTRAIL-R3: Protein array data detected sTRAIL-R3 in the
supernatant of OpRegen.RTM. cells (Tables 9 and 10). ELISA
confirmed the presence of sTRAIL-R3 along OpRegen.RTM. production
process (493 pg/mL in QC3 and 238 pg/mL in QC4). In fetal HuRPE
culture there was no sTRAIL-R3 and in RPE 7 culture, very low
levels of sTRAIL-R3 (4 pg/mL).
[0434] Detection of MIF: Protein array data detected MIF in the
supernatant of OpRegen.RTM. cells (Tables 9 and 10). ELISA
confirmed the presence of MIF along OpRegen.RTM. production process
(100.3 ng/mL in QC3 and 44.7 ng/mL in QC4). Polarized OpRegen.RTM.
cells demonstrated higher levels of MIF in the apical side (apical
MIF levels in the range of 26.6-138.3 ng/mL and basal in the range
of 1.9-30.5 ng/mL).
Example 10
Comparison of OpRegen.COPYRGT. to RPE1 & RPE7
[0435] Objective: To compare OpRegen.RTM. (RPE cells) with RPE
cells generated according to the protocol of Idelson et al,
2009.
[0436] Materials and Methods
[0437] OpRegen.RTM. (RPE cells) were generated as described in
Example 3.
[0438] RPE cells were generated according to the protocol of
Idelson et al, 2009 and named RPE1 and RPE7.
[0439] A transwell system (as illustrated in FIG. 28) was used to
enable the development of a polarized RPE monolayer with stable
barrier properties and polarized PEDF and VEGF secretion.
Transepithelial electrical resistance (TEER) measurements were used
to assess the barrier function of the RPE monolayer, and
Enzyme-Linked Immunosorbent Assay (ELISA) was used to assess
polarized PEDF and VEGF secretion. Cells were thawed and cultured
for 14 days in the presence of Nicotinamide. PEDF secretion was
tested on days 7 and 14. Then cells were transferred to a transwell
(Costar 3460, 0.4 .mu.m) for additional 4 weeks during which TEER
was measured and medium was collected (for assessment of cytokine
secretion) from the upper and lower transwell chambers on a weekly
basis up to 4 weeks. When the cells are polarized, TEER should be
above 100.OMEGA. and the ratio between the apical to basal PEDF
secretion and the basal to apical VEGF secretion should be above
1.
[0440] All OpRegen.RTM. batches that were tested demonstrated the
ability to generate barrier function (TEER range of 368-688.OMEGA.)
and secrete PEDF and VEGF in a polarized manner (Apical/Basal PEDF
ratio ranged from 3.47-8.75 and Basal/Apical VEGF ratio of
1.39-2.74) (see Table 11).
TABLE-US-00011 TABLE 11 Non-GMP GMP Mock Produced RPE Production
According to Criteria OpRegen.RTM. Clinical- OpRegen.RTM. GMP
Produced OpRegen.RTM. Idelson et al., Test for Grade Batches
Research-Grade Batches Batches 2009 Test Method release 2A 2B 6 5A
5B 5C 5D #4 #5 RPE1 RPE7 RPE Purity % AM- .gtoreq.95% 98.85% 98.26%
99.08 98.91% 99.01% 99.24% 99.29% 99.61% 99.76% 99.91% 96.29%
CRLBP.sup.+PM RPE-04 EL17.sup.+ Potency Polarization - AM- For 532
458 411 451 468 368 543 688 616 <100 <100 TEER RPE-15
informa- .OMEGA. .OMEGA. .OMEGA. .OMEGA. .OMEGA. .OMEGA. .OMEGA.
.OMEGA. .OMEGA. .OMEGA. .OMEGA. at Week 3 tion PEDF only 8.75 6.12
5.77 3.47 4.46 3.86 4.16 6.78 3.93 ND ND Apical/Basal Ratio at Week
3 VEGF 2.27 2.35 2.51 1.86 1.39 1.86 1.97 2.57 2.74 ND ND
Basal/Apical Ratio at Week 3 PEDF 3033 2158 2881 1562 1255 1551
1370 2462 3936 2279 2556 secretion day 14 (ng/ml/day) ND: Not
determined since TEER was below 100 .OMEGA. and big holes were seen
in the culture
[0441] RPE1 and RP7, that were produced under GMP conditions
according to Idelson et al (2009) were unable to generate barrier
function (TEER <100.OMEGA.) in 3 independent studies. Cells
seeded on the transwell were unable to generate a homogeneous
closed polygonal monolayer and big holes were seen (FIG. 36).
Although the cells could not generate barrier function, RPE1 and
RPE7 could secrete PEDF (see Table 11) and VEGF (not shown) in
levels similar to OpRegen.RTM. and their level of
CRALBP.sup.+PMEL17.sup.+ purity was 99.91% and 96.29%,
respectively, similar to OpRegen.RTM. (FIG. 37).
[0442] Based on these data, it may be concluded that RPE1 and RPE7
are defective in their ability to generate tight junction.
[0443] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0444] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
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