FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
1. Title of the invention: UNIFIED CELL DIFFERENTIATION PROTOCOL
2. Applicant(s)
NAME NATIONALITY ADDRESS
EYESTEM RESEARCH
PRIVATE LIMITED
Indian 106A Shagun Complex B 93 Swastik
Society Navrangpura, Ahmedabad,
380009, India
3. Preamble to the description
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it
is to be performed.
1
2
FIELD OF INVENTION
[001] The present disclosure relates to the field of cell culture techniques 5 and cell
differentiation protocols in general and cell differentiation protocol for producing
multiple retinal cells in particular.
BACKGROUND OF THE INVENTION
10
[002] Retina is a layered structure and forms the third and inner coat of the eye. It
is a light sensitive layer of tissue and comprises multiple cells with varying
functions. Photoreceptor cells are the only neurons in the retina that are directly
sensitive to light and function by converting light to signals that can stimulate
15 biological processes. Rods, cones and photosensitive retinal ganglion cells are the
three different types of photoreceptor cells found in the human eye.
[003]RPE or retinal pigmented epithelium comprises cells which interact closely
with the photoreceptors for the purpose of visual function. RPE is also responsible
for transport of nutrients, ions and water, phagocytosis of shed photoreceptor
20 membranes and for secretion of essential factors for the structural integrity of the
retina (Simo et al. Journal of Biomedicine and Biotechnology, 2010, 15 Article ID
190724).
[004] Due to their association with major complex functions, the photoreceptor
cells and the RPE cells are the most important cells of the retina and malfunctioning
25 of any of the two cells leads to development of serious eye defects. Genetic
components determine the genesis and health of photoreceptors, and mutations that
lead to structural and/or functional perturbations can eventually lead to blindness
(Veleri et al. Dis Model Mech. 2015. 8(2: 109-129).
[005] Retinal degeneration is described as the destruction or deterioration of the
30 retina caused by progressive and eventual death of the retinal cells. The disorders
3
associated with retinal degeneration are collectively termed as retinal
degenerative disorders. Diabetic retinopathy, retinopathy of prematurity, macular
degeneration, Usher syndrome, Stargardt disease and Retinis Pigmentosa are few
of the prevalent retinal degenerative diseases.
[006] WO2017091844 A1 provides a differentiation protocol to 5 generate retinal
pigmented epithelial cells for the in vitro differentiation of mammalian pluripotent
stem cells to mature, functional retinal pigment epithelial cells.
[007] US8956866 B2 describes a method of promoting directed differentiation of
human pluripotent stem cells into retinal pigment epithelium fate.
10 [008] Recent years have witnessed multiple cases of retinal degenerative disorders.
Constant efforts are being made in this field and a number of protocols have also
been designed for production of retinal cells. However, most of these methods
are time consuming and tend to be inefficient. Therefore, there is a need for
developing protocols for generating retinal cells that are not only efficient but are
15 also cost effective.
SUMMARY OF INVENTION
[007] In an aspect of the present disclosure, there is provided a method for
20 producing multiple cell types of retina from pluripotent stem cells, said method
comprising: (a) generating non-adherent suspension embryoid bodies from the
pluripotent stem cells in a medium A, wherein the medium A comprises growth
factors, organic molecules, and lipid concentrates; (b) culturing the non-adherent
suspension embryoid bodies in a medium B for 3-5 days, wherein the medium B
25 comprises at least one WNT pathway inhibitor and at least one ALK receptor
inhibitor; (c) culturing the embryoid bodies of step (b) on plates coated with at
least one suitable extracellular matrix in the medium B for 4-8 days; (d) culturing
the embryoid bodies or adherent cultures of step (c) in a medium C for 5-20
days to form neural rosette like structures and epithelial like retinal progenitor
4
cells, wherein the medium C does not comprise any inhibitor; (e) selecting the
neural rosette like structures using a selection reagent, wherein the selection
reagent allows preferential isolation and enrichment of the neural rosette like
structures; (f) culturing the neural rosette like structures of step (e) on plates coated
with at least one suitable extracellular matrix in a modified 5 medium C until
confluence for formation of photoreceptor progenitors, wherein the modified
medium C comprises at least one ROCK1 inhibitor and NOTCH inhibitor; (g)
culturing the photoreceptor progenitors in the modified medium C for 8-12 weeks
to form mature photoreceptor cells; (h) selecting and culturing the epithelial like
10 retinal progenitor cells obtained in step (d) in the medium C for 5-7 days for
formation of retinal pigmented epithelial progenitors; (i) culturing the retinal
pigmented epithelial progenitors of step (h) in a medium D for 15-45 days to obtain
mature retinal pigmented epithelial cells, wherein the medium D comprises taurine,
hydrocortisone and triido-thyronine; (j) culturing the non-adherent suspension
15 embryoid bodies of step (a) in the medium B for 8-10 days; and (k) culturing the
non-adherent suspension embryoid bodies of step (j) in the medium C for 8-12
weeks for formation of 3D retinal organoids, wherein the method is a unified method
for producing multiple cell types of retina.
[008] In an aspect of the present disclosure, there is provided a method for
20 producing multiple cell types of retina from pluripotent stem cells, said method
comprising: (a) o b t a i n i n g a d h e r e n t c u l t u r e s b y direct differentiation
from the pluripotent stem cells in a medium A, wherein the medium A
comprises growth factors, organic molecules, and lipid concentrates; (b) culturing
the adherent cultures in a medium B for 3-5 days, wherein the medium B comprises
25 at least one WNT pathway inhibitor and at least one ALK receptor inhibitor;
(c) culturing the adherent cultures of step (b) on plates coated with at least one
suitable extracellular matrix in the medium B for 4-8 days; (d) culturing the
embryoid bodies or adherent cultures of step (c) in a medium C for 5-20 days
to form neural rosette like structures and epithelial like retinal progenitor cells,
5
wherein the medium C does not comprise any inhibitor; (e) selecting the neural
rosette like structures using a selection reagent, wherein the selection reagent
allows preferential isolation and enrichment of the neural rosette like structures;
(f) culturing the neural rosette like structures of step (e) on plates coated with at
least one suitable extracellular matrix in a modified medium C until 5 confluence for
formation of photoreceptor progenitors, wherein the modified medium C comprises
at least one ROCK1 inhibitor and NOTCH inhibitor; (g) culturing the
photoreceptor progenitors in the modified medium C for 8-12 weeks to form
mature photoreceptor cells; (h) selecting and culturing the epithelial like retinal
10 progenitor cells obtained in step (d) in the medium C for 5-7 days for formation of
retinal pigmented epithelial progenitors; (i) culturing the retinal pigmented
epithelial progenitors of step (h) in a medium D for 15-45 days to obtain mature
retinal pigmented epithelial cells, wherein the medium D comprises taurine,
hydrocortisone and triido-thyronine, and wherein the method is a unified method
15 for producing multiple cell types of retina.
[009] In an aspect of the present disclosure, there is provided a pharmaceutical
composition comprising photoreceptor cells obtained from any one method as
described herein above.
[0010] In an aspect of the present disclosure, there is provided a pharmaceutical
20 composition comprising retinal pigmented epithelial cells obtained from any one
method as described herein above.
[0011] In an aspect of the present disclosure, there is provided a pharmaceutical
composition comprising 3D retinal organoids obtained from a method comprising:
(a) generating non-adherent suspension embryoid bodies from the pluripotent
25 stem cells in a medium A, wherein the medium A comprises growth factors,
organic molecules, and lipid concentrates; (b) culturing the non-adherent
suspension embryoid bodies in a medium B for 3-5 days, wherein the medium B
comprises at least one WNT pathway inhibitor and at least one ALK receptor
inhibitor; (c) culturing the embryoid bodies of step (b) on plates coated with at
6
least one suitable extracellular matrix in the medium B for 4-8 days; (d) culturing
the embryoid bodies or adherent cultures of step (c) in a medium C for 5-20
days to form neural rosette like structures and epithelial like retinal progenitor
cells, wherein the medium C does not comprise any inhibitor; (e) selecting the
neural rosette like structures using a selection reagent, wherein 5 the selection
reagent allows preferential isolation and enrichment of the neural rosette like
structures; (f) culturing the neural rosette like structures of step (e) on plates coated
with at least one suitable extracellular matrix in a modified medium C until
confluence for formation of photoreceptor progenitors, wherein the modified
10 medium C comprises at least one ROCK1 inhibitor and NOTCH inhibitor; (g)
culturing the photoreceptor progenitors in the modified medium C for 8-12 weeks
to form mature photoreceptor cells; (h) selecting and culturing the epithelial like
retinal progenitor cells obtained in step (d) in the medium C for 5-7 days for
formation of retinal pigmented epithelial progenitors; (i) culturing the retinal
15 pigmented epithelial progenitors of step (h) in a medium D for 15-45 days to obtain
mature retinal pigmented epithelial cells, wherein the medium D comprises taurine,
hydrocortisone and triido-thyronine; (j) culturing the non-adherent suspension
embryoid bodies of step (a) in the medium B for 8-10 days; and (k) culturing the
non-adherent suspension embryoid bodies of step (j) in the medium C for 8-12
20 weeks for formation of 3D retinal organoids, wherein the method is a unified method
for producing multiple cell types of retina..
[0012] These and other features, aspects, and advantages of the present subject
matter will be better understood with reference to the following description and
appended claims. This summary is provided to introduce a selection of concepts in
25 a simplified form. This summary is not intended to identify key features or
essential features of the claimed subject matter, nor is it intended to be used to
limit the scope of the claimed subject matter.
7
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0013] The following drawings form a part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may
be better understood by reference to the drawings in combination 5 with the detailed
description of the specific embodiments presented herein.
[0014] Figures 1A-1F shows phase contrast images displaying temporal changes in
cellular morphology from undifferentiated iPSCs to photoreceptor-like cells, in
accordance with an embodiment of the present disclosure.
10 [0015] Figures 2A-2I depicts immunostaining of PR cells with different cell
biomarkers, in accordance with an embodiment of the present disclosure.
[0016] Figures 3A-3F depicts results of gene expression profile of different cell
biomarkers of PR cells on differentiation days 7, 21, 49, and 77, in accordance with
an embodiment of the present disclosure.
15 [0017] Figures 4A-4L depicts phase contrast microscopy and immunofluorescence
analysis of RPE differentiation, in accordance with an embodiment of the present
disclosure.
[0018] Figures 5A-5L depicts authentication of RPE by immunohistochemistry
studies, in accordance with an embodiment of the present disclosure.
20 [0019] Figures 6A-6D depicts quantification of RPE cells with respect to gene
expression profile and biomarkers, in accordance with an embodiment of the present
disclosure.
[0020] Figures 7A and 7B display phase contrast images of organoids at day 60,
Figure 7C and 7D displays immunostaining of 10μm sections of 3D retinal
25 organoid for PR marker and RPE marker, in accordance with an embodiment of
the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
8
Those skilled in the art will be aware that the present disclosure is subject to
variations and modifications other than those specifically described. It is to be
understood that the present disclosure includes all such variations and
modifications. The disclosure also includes all such steps, features,
compositions, and compounds referred to or indicated in this 5 specification,
individually or collectively, and any and all combinations of any or more of such
steps or features.
Definitions
[0021] For convenience, before further description of the present disclosure, certain
10 terms employed in the specification, and examples are delineated here. These
definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the
meanings recognized and known to those of skill in the art, however, for
convenience and completeness, particular terms and their meanings are set forth
15 below.
[0022] The articles “a”, “an” and “the” are used to refer to one or to more than
one (i.e., to at least one) of the grammatical object of the article.
[0023] The terms “comprise” and “comprising” are used in the inclusive, open
sense, meaning that additional elements may be included. It is not intended to be
20 construed as “consists of only”.
[0024] Throughout this specification, unless the context requires otherwise the
word “comprise”, and variations such as “comprises” and “comprising”, will be
understood to imply the inclusion of a stated element or step or group of element or
steps but not the exclusion of any other element or step or group of element or steps.
25 [0025] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
[0026] Pluripotent stem cells are stem cells that has the potential to differentiate
into any of the three germ layers: endoderm, mesoderm or ectoderm.
9
[0027] Xenofree refers to products that do not contain any animal derived
component.
[0028] Retinal Pigmented Epithelium (RPE) is the pigmented cell layer just
outside the neurosensory retina that nourishes retinal visual cells.
[0029] Photoreceptor cell is a specialized cell found in the 5 retina. The cells
are responsible for visual phototransduction.
[0030] Adherent cells are the cells that are anchorage dependent and have to
be cultured on a suitable substrate that is specifically treated to promote cell
adhesion and growth.
10 [0031] Unless defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in the
art to which this disclosure belongs. Although any methods and materials similar
or equivalent to those described herein can be used in the practice or testing of the
disclosure, the preferred methods, and materials are now described. All
15 publications mentioned herein are incorporated herein by reference.
[0032] Abbreviations:
1.EDTA: Ethylenediaminetetraacetic acid
2. DMEM/F12: Dulbecco's Modified Eagle's medium/F12
3. ALK: Anaplastic lymphoma kinase
20 4.ROCK1: Rho Kinase
5. WNT: wingless-type MMTV
6. IGF: insulin like growth factor
7. VTN: Vitronectin
8. MEM/alpha: Modified Eagle’s medium alpha
25 9. KOSR: Knockout serum
10.NEAA: Non-essential amino acids
11.ELISA: Enzyme linked immunosorbent assay
12. DPBS: Dulbecco's phosphate-buffered saline
13. WNT: Refers to Wnt signalling pathway
10
14. NOTCH: Refers to Notch signalling pathway
[0033] Retinal degenerative diseases are caused due to deterioration of retinal
cells and tissues and leads to progressive and eventual death of the cells of retina.
Depending upon the severity of the disease it can result in 5 varying degrees of
irreversible vision loss. Currently there are millions of people worldwide
who are suffering from different retinal degenerative disorders. One of the most
common retinal degenerative diseases in terms of its occurrence worldwide is
macular degeneration or age related macular degeneration. Macula is the part of
10 the eye that helps in fine-tuning the details and is responsible for the central
vision. Degeneration of the macula results in blurred or no vision in the centre of the
visual field. There are supplements and medicaments, which are available for
slowing down the progression of the disease, however they are not effective for
preventing the disorder. Stem cell or retinal cell transplants have emerged as an
15 effective therapy against the degenerative diseases. Advancements in the field
have led to embryonic stem cell trials using human stem cell derived RPEs
(Schwartz et al. Lancet. 2012. 25;379(9817): 713-20).
[0034] The major limitation in the cell transplant based therapy is generation
of retinal cells in a cost effective and efficient manner. Various protocols and
20 methods have been explored for producing retinal cells such as photoreceptors,
and retinal pigment epithelial cells. However, most of these methods are constrained
by time and efficiency limitation.
[0035] The present disclosure provides with a method for generating multiple
cell types of retina by differentiating induced pluripotent stem cells (iPSCs). The
25 disclosure reveals a unified method, which can be employed for production of
photoreceptors, retinal pigmented epithelial cells and 3D retinal organoids from a
single source of pluripotent stem cells. The protocol describes generation of
suspension embryoid bodies from iPSCs which can further be grown under
specified and optimized conditions for generation of photoreceptors, retinal
11
pigmented epithelial cells and 3D retinal organoids. The protocol as described in
the present disclosure also discloses generation of adherent cultures from iPSCs
which can further be grown under specified and optimized conditions for generation
of photoreceptors, retinal pigmented epithelial cells and 3D retinal organoids. The
protocol employs media compositions comprising growth factors, 5 inducers and
inhibitors that lead to differentiation of embryoid bodies into desired cell types.
[0036] The present disclosure is not to be limited in scope by the specific
embodiments described herein, which are intended for the purposes of
exemplification only. Functionally-equivalent products, compositions, and
10 methods are clearly within the scope of the disclosure, as described herein.
[0037] In an embodiment of the present disclosure, there is provided a
method for producing multiple cell types of retina from pluripotent stem cells, said
method comprising: (a) generating non-adherent suspension embryoid bodies
from the pluripotent stem cells in a medium A, wherein the medium A comprises
15 growth factors, organic molecules, and lipid concentrates; (b) culturing the nonadherent
suspension embryoid bodies in a medium B for 3-5 days, wherein the
medium B comprises at least one WNT pathway inhibitor and at least one
ALK receptor inhibitor; (c) culturing the embryoid bodies of step (b) on plates
coated with at least one suitable extracellular matrix in the medium B for 4-7
20 days; (d) culturing the embryoid bodies of step (c) in a medium C for 5-10 days to
form neural rosette like structures and epithelial like retinal progenitor cells,
wherein the medium C does not comprise any inhibitor; (e) selecting the neural
rosette like structures using a selection reagent, wherein the selection reagent allows
preferential isolation and enrichment of the neural rosette like structures; (f)
25 culturing the neural rosette like structures of step (e) on plates coated with at least
one suitable extracellular matrix in a modified medium C until confluence for
formation of photoreceptor progenitors, wherein the modified medium C
comprises at least one ROCK inhibitor and at least one NOTCH inhibitor; (g)
culturing the photoreceptor progenitors in the modified medium C for 8-12 weeks
12
to form mature photoreceptor cells; (h) selecting and culturing the epithelial like
retinal progenitor cells obtained in step (d) in the medium C for 5-7 days for
formation of retinal pigmented epithelial progenitors; (i) culturing the retinal
pigmented epithelial progenitors of step (h) in a medium D for 8 -10 days
to obtain mature retinal pigmented epithelial cells, wherein 5 the medium D
comprises taurine, hydrocortisone and triido-thyronine; (j) culturing the nonadherent
suspension embryoid bodies of step (a) in the medium B for 8-10 days;
and (k) culturing the non-adherent suspension embryoid bodies of step (j) in the
medium C for 8-12 weeks for formation of 3D retinal organoids, wherein the
10 method is a unified method for producing multiple cell types of the retina, to obtain
multiple cell types of retina from pluripotent stem cells.
[0038] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells, said method
comprising: (a) obtaining adherent cultures by direct differentiation from the
15 pluripotent stem cells in a medium A, wherein the medium A comprises growth
factors, organic molecules, and lipid concentrates; (b) culturing the adherent
cultures in a medium B for 3-5 days, wherein the medium B comprises at least one
WNT pathway inhibitor and at least one ALK receptor inhibitor; (c) culturing
the adherent cultures of step (b) on plates coated with at least one suitable
20 extracellular matrix in the medium B for 4-7 days; (d) culturing the adherent
cultures of step (c) in a medium C for 5-20 days to form neural rosette like structures
and epithelial like retinal progenitor cells, wherein the medium C does not comprise
any inhibitor; (e) selecting the neural rosette like structures using a selection reagent,
wherein the selection reagent allows preferential isolation and enrichment of the
25 neural rosette like structures; (f) culturing the neural rosette like structures of step
(e) on plates coated with at least one suitable extracellular matrix in a modified
medium C until confluence for formation of photoreceptor progenitors, wherein the
modified medium C comprises at least one ROCK inhibitor, and at least one
NOTCH inhibitor; (g) culturing the photoreceptor progenitors in the modified
13
medium C for 8-12 weeks to form mature photoreceptor cells; (h) selecting and
culturing the epithelial like retinal progenitor cells obtained in step (d) in the
medium C for 5-7 days for formation of retinal pigmented epithelial progenitors; (i)
culturing the retinal pigmented epithelial progenitors of step (h) in a medium D
for 8 -10 days to obtain mature retinal pigmented epithelial 5 cells, wherein the
medium D comprises taurine, hydrocortisone and triido-thyronine, to obtain
multiple cell types of retina from pluripotent stem cells.
[0039] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as described
10 herein, wherein selecting the neural rosette like structures is alternatively done by
manual picking.
[0040] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as described
herein, wherein the multiple cell types of retina comprise photoreceptor cells,
15 retinal pigmented epithelial cells and 3D retinal organoids.
[0041] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as described
herein, wherein the pluripotent stem cells are induced pluripotent stem cells.
[0042] In an embodiment o f the present disclosure, there i s provided a
20 method for producin g multipl e cell types of retin a from pluripoten t stem
cells as described herein, wherein generating the non-adherent suspension
embryoid bodies from the pluripotent stem cell comprises: (a) growing the
pluripotent stem cells on a plate coated with at least one suitable extracellular
matrix in a medium A until confluence, wherein the medium A comprises
25 growth factors, organic molecules and lipid concentrates; (b) dislodging the
pluripotent stem cells of step (a) using a cell detachment enzyme solution
to obtain detached pluripotent stem cells; (c) neutralizing the detached
pluripotent stem cells with a neutralization medium to obtain neutralized
14
pluripotent stem cell, wherein the neutralization medium comprises 5%-20% KOSR
or a suitable neutralizing agent; and (d) seeding the neutralized pluripotent
stem cells on a non-adherent suspension culture plate in a modified medium
A for generating the non- adherent suspension embryoid bodies, wherein the
modified medium A comprises at least one ROCK inhibitor. 5 In another
embodiment, the non-adherent suspension embryoid bodies generated in step (d) is
further maintained in the modified medium A for 24-48 hour.
[0043] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as described
10 herein, wherein generating the adherent cell comprises: (a) growing the pluripotent
stem cells on a plate coated with at least one suitable extracellular matrix in a
medium A until confluence, wherein the medium A comprises growth factors,
organic molecules and lipid concentrates.
[0044] In an embodiment of the present disclosure, there is provided a method
15 for producing multiple cell types of retina from pluripotent stem cells as
described herein, wherein the mature retinal pigmented epithelial cells obtained
in step (i) is further passaged, comprising: (a) incubating the retinal
pigmented epithelial cells with 0.05% - 0.5% trypsin EDTA solution for
detachment of non-pigmented and non-retinal pigmented epithelial cells for 2-10
20 minutes; (b) discarding the non-pigmented and non-retinal pigmented epithelial
cells to obtain a pure population of retinal pigmented epithelial cells; (c) incubating
the pure population of retinal pigmented epithelial cells with medium E, wherein
the medium E comprises accutase enzyme and 0.2-0.5% trypsin EDTA; (d)
neutralizing the pure population of retinal pigmented epithelial cells of step (c) using
25 neutralization medium comprising 5-20% KOSR; and (e) culturing the neutralized
pure population of retinal pigmented cells on a plate coated with at least one suitable
extracellular matrix in the medium D. In another embodiment of the present
disclosure, the retinal pigmented cells are alternatively incubated in medium E,
15
wherein the medium E alternatively comprises at least one substance selected from
a group consisting of collagenase enzyme, accustase, TrypLE Express, TrypLE
Select, and compositions thereof, and wherein the neutralization medium
alternatively comprises any suitable neutralizing agent well known in the art. In yet
another embodiment of the present disclosure, wherein 5 detachment of nonpigmented
and non-retinal pigmented epithelial cells is done by manual selection
under stereomicroscope.
[0045] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as
10 described herein, wherein the at least one WNT inhibitor is selected from the group
consisting of 4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-
yl)-N-8- quinolinyl-Benzamide, 5-(Phenylsulfonyl)-N-piperidin-4-yl-2-
(trifluoromethyl) benzene sulfonamide, 2-(2',3-Dimethyl-[2,4'-bipyridin]-5-
yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl) acetamide, 2-(4-(2-methylpyridin-4-
15 yl)phenyl)-N-(4-(pyridin-3- yl)phenyl) acetamide, 8-Tetrahydro-2-[4-
(trifluoromethyl)phenyl]-4H- thiopyrano[4,3-d]pyrimidin-4-one, and combinations
thereof.
[0046] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as described
20 herein, wherein the at least one WNT inhibitor is 4-(1,3,3a,4,7,7a-Hexahydro-
1,3- dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide.
[0047] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as
described herein, wherein the at least one ALK receptor inhibitor is selected from
25 the group consisting of 4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1Himidazol-
2-yl] benzamide, 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-
a]pyrimidin-3-yl)quinoline, 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-
piperidin-4-ylpyrazol-4- yl)pyridin-2-amine, 5-chloro-2-N-[2-methoxy-4-[4-
(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]-4-N-(2-propan-2-
16
ylsulfonylphenyl)pyrimidine-2,4-diamine, and 9-ethyl-6,6-dimethyl-8-(4-
morpholin-4-ylpiperidin-1-yl)-11-oxo-5H- benzo[b]carbazole-3-carbonitrile, 5-
chloro-2-N-(5-methyl-4-piperidin-4-yl-2-propan-2-yloxyphenyl)-4-N-(2-propan-2-
ylsulfonylphenyl) pyrimidine-2,4-diamine, and combinations thereof.
[0048] In an embodiment of the present disclosure, there is 5 provided a method
for producing multiple cell types of retina from pluripotent stem cells as described
herein, wherein the at least one ALK receptor inhibitor is either 4-[4-(1,3-
benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl] benzamide or 4-(6-(4-
(piperazin-1-yl) phenyl) pyrazolo[1,5-a]pyrimidin-3-yl)quinoline. In another
10 embodiment, the at least one ALK inhibitor is a combination of 4-[4-(1,3-
benzodioxol-5-yl)-5-pyridin-2- yl-1H-imidazol-2-yl] benzamide and 4-(6-(4-
(piperazin-1-yl) phenyl) pyrazolo[1,5- a]pyrimidin-3-yl)quinoline.
[0049] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as
15 described herein, wherein the at least one NOTCH inhibitor is selected from a group
consisting of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl
ester (DAPT), N-[cis-4-[(4-Chlorophenyl)sulfonyl]-4-(2,5-
difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide, 2-[(1R)-1-[[(4-
Chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-fluorobenzenebutanoic
20 acid, 5-Chloro-N-[(1S)-3,3,3-trifluoro-1-(hydroxymethyl)-2-
(trifluoromethyl)propyl]-2-thiophenesulfonamide, N-[(1S)-2-[[(7S)-6,7-Dihydro-
5-methyl-6-oxo-5H-dibenz[b,d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-
difluorobenzeneacetamide, (R)-2-Fluoro-α-methyl[1,1'-biphenyl]-4-acetic acid, N-
[(1S)-2-[[(3S)-2,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-
25 yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide, 3,5-Bis(4-
nitrophenoxy)benzoic acid, (5S)-(tert-Butoxycarbonylamino)-6-phenyl-(4R)-
hydroxy-(2R)-benzylhexanoyl)-L-leucy-L-phenylalaninamide, 7-Amino-4-chloro-
3-methoxy-1H-2-benzopyran, (2S)-N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-
phenyl]glycine 1,1-dimethylethyl ester, and combinations thereof. In another
17
embodiment of the present disclosure, the at least NOTCH inhibitor is N-[N-(3,5-
Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).
[0050] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as described
herein, wherein the at least one suitable extracellular matrix 5 is selected from
the group consisting of l ami n i n , vitronectin, fibronectin, collagen, poly-Llysine,
poly-L-ornithine, and combinations thereof. In another embodiment, the at
least one suitable extracellular matrix is vitronectin.
[0051] In an embodiment of the present disclosure, there is provided a method
10 for producing multiple cell types of retina from pluripotent stem cells as described
herein, wherein the at least one suitable extracellular matrix is xenofree.
[0052] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as described
herein, wherein the medium C further comprises DMEM/F12, KOSR, and NEAA.
15 [0053] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as
described herein, wherein the at least one ROCK inhibitor is selected from
the group consisting of 4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-
carboxamide, 4- [(1R)-1-aminoethyl]-N-(1H-pyrrolo[2,3-b] pyridin-4-
20 yl)benzamide, 6- aminochromen-4-one, 5-(1,4-diazepan-1-
ylsulfonyl)isoquinoline, 4-methyl-5-[[(2S)-2-methyl-1,4-diazepan-1-
yl]sulfonyl]isoquinoline, and combinations thereof.
[0054] In an embodiment of the present disclosure, there is provided a method
for producing multiple cell types of retina from pluripotent stem cells as described
25 herein, wherein the at least one ROCK inhibitor is 4-[(1R)-1-aminoethyl]-Npyridin-
4-ylcyclohexane-1-carboxamide.
[0055] In an embodiment of the present disclosure, there is provided
photoreceptor cells produced from the method as described herein.
18
[0056] In an embodiment of the present disclosure, there is provided retinal
pigmented epithelial cells produced from the method as described herein.
[0057] In an embodiment of the present disclosure, there is provided 3D retinal
organoids produced from the method as described herein.
[0058] In an embodiment of the present disclosure, there is provided 5 3D optic cups
produced from the method as described herein.
[0059] In an embodiment of the present disclosure, there is provided a
pharmaceutical composition comprising the photoreceptor cells produced from
the method as described herein.
10 [0060] In an embodiment of the present disclosure, there is provided a
pharmaceutical composition comprising the retinal pigmented epithelial cells
produced from the method as described herein.
[0061] In an embodiment of the present disclosure, there is provided a
pharmaceutical composition comprising the 3D retinal organoids produced from
15 the method as described herein.
[0062] In an embodiment of the present disclosure, there is provided a
pharmaceutical composition comprising the photoreceptor cells produced from
the method as described herein, wherein the composition is used in manufacture
of a medicament.
20 [0063] In an embodiment of the present disclosure, there is provided a
pharmaceutical composition comprising the retinal pigmented epithelial cells
produced from the method as described herein, wherein the composition is used
in manufacture of a medicament.
[0064] In an embodiment of the present disclosure, there is provided a
25 pharmaceutical composition comprising the 3D retinal organoids produced from
the method as described herein, wherein the composition is used in manufacture of
a medicament.
[0065] In an embodiment of the present disclosure, there is provided a
pharmaceutical composition comprising the photoreceptor cells produced from
19
the method as described herein, wherein the composition is used in manufacture of
a medicament, and wherein the medicament is used for treatment of retinal
degenerative diseases.
[0066] In an embodiment of the present disclosure, there is provided a
pharmaceutical composition comprising the retinal pigmented 5 epithelial cells
produced from the method as described herein, wherein the composition is used
in manufacture of a medicament, and wherein the medicament is used for treatment
of retinal degenerative diseases.
[0067] In an embodiment of the present disclosure, there is provided a
10 pharmaceutical composition comprising the 3D retinal organoids produced from
the method as described herein, wherein the composition is used in manufacture of
medicament, and wherein the medicament is used for treatment of retinal
degenerative diseases.
[0068] In an embodiment of the present disclosure, there is provided a
15 pharmaceutical composition comprising the photoreceptor cells produced from
the method as described herein, wherein the composition is used in manufacture of
a medicament, and wherein the medicament is used for treatment of
retinal degenerative diseases selected from the group consisting of retinitis
pigmentosa, Best disease, Stargardt disease, Usher syndrome, rod-cone
20 dystrophy, and age related macular degeneration.
[0069] In an embodiment of the present disclosure, there is provided
a pharmaceutical composition comprising the retinal pigmented epithelial
cells produced from the method as described herein, wherein the composition is
used in manufacture of a medicament, and wherein the medicament is used for
25 treatment of retinal degenerative diseases selected from the group consisting of
retinitis pigmentosa, Best disease, Stargardt disease, Usher syndrome, rod-cone
dystrophy, and age related macular degeneration..
[0070] In an embodiment of the present disclosure, there is provided
pharmaceutical composition comprising the 3D retinal organoids produced from
20
the method as described herein, wherein the composition is used in manufacture of
a medicament, and wherein the medicament is used for treatment of
retinal degenerative diseases selected from the group consisting of retinitis
pigmentosa, Best disease, Stargardt disease, Usher syndrome, rod-cone
dystrophy, and age related macular 5 degeneration.
EXAMPLES
[0071] The disclosure will now be illustrated with working examples, which is
intended to illustrate the working of disclosure and not intended to take
10 restrictively to imply any limitations on the scope of the present disclosure. Unless
defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood to one of ordinary skill in the art to which this
disclosure belongs. Although methods and materials similar or equivalent to
those described herein can be used in the practice of the disclosed methods and
15 compositions, the exemplary methods, devices and materials are described herein.
It is to be understood that this disclosure is not limited to particular methods, and
experimental conditions described, as such methods and conditions may vary.
[0072] The examples below describe the protocol disclosed in the present invention
in detail. The protocol focuses on the differentiation of induced pluripotent stem cell,
20 to form multiple cell types of retina. However, it should be noted that the present
protocol can be used for differentiating other pluripotent stem cells as well, such
as, embryonic stem cells. Apart from providing with a unified protocol for
generating multiple cell types of retina, the protocol also provides with method
for generating suspension type embryoid bodies from pluripotent stem cell. The
25 suspension type embryoid bodies are further cultured under specific and optimized
media and incubation conditions for formation of retinal cells such as
photoreceptors and RPEs.
[0073] The cells obtained from the protocol were further verified by using
immunofluorescence and RT-PCR technique. The cells display expression of
21
important and relevant markers with respect to the days of differentiation. The
features that differentiate the current protocols from other similar cell culture
protocols include the media composition, number of days required for cell
differentiation, selection of neural rosette like structures using non-enzyme based
selection reagent or manual picking of rosette like structures, 5 which allows
preferential isolation and enrichment of the neural rosettes.
Materials and Methods
[0074] Composition of the different media as used in the protocol is described
10 below:
Composition of Medium A- Medium A refers to mTESR medium procured
form Stem Cell Technologies or Stemflex or E8 medium procured from
ThermoFisher Scientific. Modified medium A has an additional supplementary
component as compound 4-[(1R)-1-aminoethyl]-N-pyridin-4-
15 ylcyclohexane-1- carboxamide (Y27632).
[0075] Composition of Medium B- Medium B here refers to Differentiation
Induction Medium (DIM) having composition as below:
[0076] Table 1:
Components For 50 ml Final concentration
DMEM/F12 42.5ml 10%
KOSR 5ml 1%
Sodium pyruvate 0.5ml 1%
Sodium bicarbonate 0.5ml 1%
HEPES buffer 0.5ml 1%
Non-essential amino acids 0.5ml 1%
N1 media supplements 0.5ml 1X
Compound 1 (2mM) * 50μl 2μM
Compound 2 (10mM)* 50μl 10μM
22
Compound 3 (1mM) * 5μl 100nM
IGF1 (100ng/ml) 5μl 10ng/ml
Activin A 10μl 10μg/ml
Nicotinamide 5μl 10mM
[0077] *Compound 1: 4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2Hisoindol-
2- yl)-N-8-quinolinyl-Benzamide (WNT pathway inhibitor)
[0078] Compound 2: 4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1Himidazol-
2-yl]benzamide (ALK receptor inhibitor) Compound 3: 4-(6-(4-
(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline 5 (ALK receptor
inhibitor)
[0079] Composition of Medium C: Medium C here refers to differentiation
propagation medium (DPM) having composition as below:
Table 2:
10
Components For 50ml Final Concentration
DMEM/F12 47ml
Knock-Out Serum Replacement
(KOSR)
0.5ml 1%
Sodium pyruvate 0.5ml 1%
Sodium bicarbonate 0.5ml 1%
HEPES buffer 0.5ml 1%
Non-essential amino acids 0.5ml 1%
N1 media supplement (100X) 0.5ml 1X
[0080] Modified Medium C has an additional component as compound 4-[(1R)-
1- aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide (Y27632), N-[N-(3,5-
Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a NOTCH
pathway inhibitor as Compound 5 and 3-[4-Methyl-2-(2-oxo-1,2-dihydro-indol-3-
23
ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid (SU5402), a FGFR antagonist as
Compound 6.
[0081] Table 3 as mentioned below refers to Medium D (RPE maturation medium):
Table 3:
5
Components For 50ml Final Concentration
MEMα modified 46.5ml
KOSR 2.5ml 5%
Glutamax 0.5ml 1%
Taurine (50mg/ml) 0.25ml 0.25 mg/ml
Hydrocortisone (20mg/ml) 25μl 10ug/ml
Triido-Thyronine (2mg/ml) 1μl 0.0065ug/ml
N1 media supplement (100X) 0.5ml 1X
[0082] The neutralization medium used in the present study is DMEM/F12
(GIBCO) supplemented with 10% KOSR or a suitable neutralization agent.
[0083] The selection reagent used in the present study is Stem Diff Neural rosette
selection reagent from Stem Cell Technologies.
[0084] Table 4 below provides different reagents and the 10 vendors they were
procured from: Table 4:
Chemicals Company Catalog No
Vitronectin ThermoFisher A14700
DMEM/F12 GIBCO 10565-018
DPBS GIBCO 14190-136
Knock out serum (KOSR) GIBCO 10828-028
HEPES buffer GIBCO 15630-080
Non-essential Amino acids GIBCO 11140-050
Accutase GIBCO A11105-01
24
Penicillin Streptomycin GIBCO 15140-122
Trypsin EDTA GIBCO 25200-072
Cryostor CS10 Stem technologies 7931
IWR1 (compound 1) Sigma I0161-25MG
N1 supplement Sigma N6530-5ML
IGF-1 Stem lltechnologies 78022
Stemflex ThermoFisher A3349401
Y27632 (ROCK 1
inhibitor)
Stem cell technologies 72302
LDN193189 (compound 2) Stem cell technologies 72142
SB431542 (compound 3) Stem cell technologies 72234
Sodium bicarbonate Thermo Fisher 25080-060
Sodium pyruvate Thermo Fisher 11360-070
Y-27632 (ROCK inhibitor) Tocris 1254
DAPT (NOTCH inhibitor,
compound 5)
Sigma D5942
SU5402 (FGFR antagonist,
compound 6)
Sigma SMLO443
Activin A ThermoFisher PHG9014
Nicotinamide Sigma 72340
TrypLE Select ThermoFisher 12563011
[0085] Differentiation protocol in detail:
Example 1
Generation of embryoid bodies: (a) Vitronectin was resuspended in DPBS at 1-
5%, added to tissue culture plates and incubated overnight to obtain
Vitronectin (VTN) coated plates. iPSCs for the present study 5 were obtained
from the umbilical cord blood of humans. Lymphocytes were isolated from the
blood and reprogrammed into iPSCs using transcription factors such as Oct4, Sox25
2, KLF4 and cMYC via episomal vectors (non-integrative). The NcGMP1
induced pluripotent stem cells (iPSC) were then grown on the VTN coated
plates in medium A and passaged regularly (passaging ratio 1:5-1:6) upon 80-90%
confluency: (b) On attaining about 80% confluence, the cells were treated with
pre-warmed dissociation enzyme (accutase) at 1ml/10cm2 and placed 5 in incubator
for 3 minutes. The enzyme was neutralized with 3 times (v:v) 10% KOSR
containing Medium D (here refers to RPE maturation DMEM-high glucose
media), and the dissociated cells were gently transferred to 15ml Falcon tube using
2 ml serological pipette avoiding dissociation of the iPSC colonies; (c) the cells were
10 centrifuged at 800g for 2 mins to form a cell pellet. After aspirating the spent
media, the cell pellet was mildly dislodged (careful so as to avoid making single
cells) and seeded onto non-adherent suspension culture plates forming forced
aggregates of embryoid bodies (EB) in modified medium A. For no n- ad he rent
cu l tur e ba s ed r et ina l di f f e r en t i a t ion th e iPSCs we r e a l lowed to
15 grow und e r s t a t i c cul tur e co ndi t io ns . The cells were maintained in
modified medium A for 24-48 hours with media change within 24-hour interval; (d)
The EBs or adherent cultures were then shifted gradually to medium B in
suspension and media was changed every day for 3-5 days. EBs were then further
used for generation of photoreceptor cells, retinal pigmented epithelial cells and 3D
20 retinal organoids, while only photoreceptor cells and retinal pigmented cells can be
generated with adherent cells.
Example 2
Generation of 3D organoids from EBs: The suspension type EBs of step (d) in
25 Example 1 were grown for another 7 days. Post 7 days, EBs were then transferred
in medium C and cultured as suspension for 12 weeks with alternate day media
change to form 3D retinal organoids or 3D optic cups.
Example 3
26
[0086] Generation of photoreceptors: The suspension type EBs or adherent
cultures of step (d) in Example 1 were transferred on 2% VTN (in medium B)
coated tissue culture plates to form adherent EBs. The cells were cultured in
modified medium B for 2 more days with media change every day. After 4-7 days,
the cells were transferred to Medium C. Upon confluency between 5 5-10 days in
Medium C, neural rosette like structures were selected using Stem Diff Neural
rosette selection reagent. The neural rosette like structures were collected with or
without dissociation using accutase enzyme. The cells were then plated on 2% VTN
coated 12 well dish in modified Medium C and cultured till they attained
10 confluency to obtain photoreceptor progenitors. The PR progenitors were further
grown and expanded in Medium C and passaged for 12 weeks to obtain
photoreceptor cells. Stock vials of PR cells can be prepared at desired day point for
further studies.
15 Example 4
[0087] Generation of RPEs: The remaining cells (epithelial like retinal progenitor
cells) that were left in the parent plate in Example 2, post selection of neural rosette
like structures were re-plated and continued for 5-7 days in Medium C to form
retinal pigmented epithelial progenitors.
20 [0088] The cells were then transferred to Medium D and grown for another 30 days
to form matured retinal epithelial cells. Stock vials of matured RPE cells were
prepared at desired day point for further studies.
Example 5
25 [0089] The RPE cells whenever confluent were split and passaged at 1:2 to 1:5
ratio by the following method:
a. 0.005-0.5% Trypsin-EDTA was added to the confluent cells and incubated at
culture conditions for 5 mins.
27
b. Non-pigmented and non RPE cells would detach at this condition and the
media with non-desired cell population was discarded.
c. Cells were replenished with TrypLE Select and or accutase and incubated at
culture conditions for 5-10 mins.
d. Cells could also be picked manually under the 5 stereomicroscope.
e. Colonies were scraped with cell lifter and clumps were dissociated in 10%
KOSR media.
f. The dissociated cells were transferred in falcon tube and centrifuged at 800g for
2 mins followed by replating on freshly coated VTN plates and further grown for
10 few passages.
Example 6
[0090] Once the cells were produced using the above unified protocol, they
were examined for expression of relevant markers.
15 [0091] Phase contrast microscopy: Figure 1 depicts the phase contrast images
of photoreceptor cells at different days throughout the differentiation. The images
display temporal changes in cellular morphology from undifferentiated iPSCs
to photoreceptor-like cells. The images are labelled as A) differentiation day 7, B)
differentiation day 21, C) differentiation day 35, D) differentiation day 49, E)
20 differentiation Day 63, and F) differentiation day 77. Gradual change in cells
differentiating form iPSCs to PR cells can be observed.
[0092] Figure 2A- 2 I depict the images of PR cells post staining with relevant
antibodies. As can be observed from the images the cells display markers, A)
Pax6, B) Sox2, C) RX, D) Thrβ, E) Otx2, F) BRN3, G) Recoverin, H) Rhodopsin,
25 and I) Nrl2
[0093] Real Time PCR quantification: Gene expression profile during
differentiation of PR cells was performed. For quantitative polymerase chain
reaction (Q-PCR) analysis, RNA was extracted by RNeasy kit (Qiagen,
Hilden, Germany, http://www.qiagen.com), and cDNA conversion was done using
28
Revert Aid kit (Thermo Scientific) as per the manufacturer’s instructions. 1 μg of
RNA was used for cDNA conversion. Q-PCR was done with SYBER Green master
mix (Life Technologies) using mRNA specific primers (sequences of primers as
listed below as Table 5) on ABI 7900HT (Life Technologies) and was analysed by
5 SDS 2.4 software.
Table 5 below lists out the sequences of the primers used in the present disclosure.
Table 5:
hPAX6 F 5’-AGTTCTTCGCAACCTGGCTA-3’
hPAX6 R 5’-TGGTATTCTCTCCCCCTCCT-3’
hZO-1 F 5’-TGAGGCAGCTCACATAATGC-3’
hZO-1 R 5’-GGGAGTTGGGGTTCATAGGT-3’
hSIX3 F 5’-CCGGAAGAGTTGTCCATGTT-3’
hSIX3 R 5’-CGACTCGTGTTTGTTGATGG-3’
hLHX2 F 5’-GCTCGGGACTTGGTTTATCA-3’
hLHX2 R 5’-GTTGAAGTGTGCGGGGTACT-3’
hRX F 5’-GAACAGCCCAAGAAAAAGCA-3’
hRX R 5’-GCTGTACACGTCCGGGTAGT-3’
hMITF F 5’-CCAGGCATGAACACACATTC-3’
hMITF R 5’-TCCATCAAGCCCAAGATTTC-3’
hRPE65 F 5’-GATCTCTGCTGCTGGAAAGG-3’
hRPE65 R 5’-TGGGGAGCGTGACTAAATTC-3’
hBESTROPHIN F 5’-GGCAGAACACAAGCAGTTGG-3’
hBESTROPHIN R 5’-ACGCAAGGTGTTCATCTCGT-3’
hTYROSINASE F 5’-ACCCATTGGACATAACCGGG-3’
29
hTYROSINASE R 5’-AGAGTCTGGGTCTGAATCTTGT-3’
hEZRIN F 5’-TCAATGTCCGAGTTACCACCA-3’
hEZRIN R 5’-GGCCAAAGTACCACACTTCC-3’
hDCT F 5’-GGTTCCTTTCTTCCCTCCAG-3’
hDCT R 5’-AACCAAAGCCACCAGTGTTC-3’
hNrl F 5’-ATG TGG ATT GGA CGA CTT C-3’
hNrl R 5’-TTG GCG AGA TTG TCT TGG-3’
hTRB2 F 5’-ACA GGA GAT TTC ATT CGG G-3’
hTRB2 R 5’-TTG TAA GAC TAT CAT CTG GGT G-3’
hBLIMP1 F 5’-GTG GTA TTG TCG GGA CTT TG-3’
hBLIMP1 R 5’-GGT TGC TTT AGA CTG CTC TG-3’
hCHX10 F 5’-CGA CAC AGG ACA ATC TTT ACC-3’
hCHX10 R 5’-CAT AGA CGT CTG GGT AGT GG-3’
hBRN3 F 5’-CTCGCTCGAAGCCTACTTTG-3’
hBRN3 R 5’-GACGCGCACCACGTTTTTC-3’
hSOX-2 F 5’-CCGCGTCAAGCGGCCCATGAA-3’
hSOX-2 R 5’-GCCGCTTCTCCGTCTCCGACAA-3’
hβ-actin F 5’-TCACCCACACTGTGCCCATCTACGA-3’
hβ-actin R 5’-CAGCGGAACCGCTCATTGCCAATGG-3’
[0094] Normalization was performed based on the average of expression of
constitutive gene β-actin. Study was performed for differentiation day 7, day 21,
day 49 and day 77. The different genes analysed are as below:
30
(a) Pan neural markers Pax-6 and Sox-2 along with early retinal marker RX: Figure
3A displays the graph depicting the gene expression profile for Pax-6. Sox-2 and
RX.
Gradual increase in expression of genes can be observed from differentiation day 7
5 to day 77.
(b) Retinal ganglion transcription factors Brn3 and CHX10: Figure 3B displays
the graph depicting the gene expression profile for transcription factors Brn3 and
CHX10. Enhancement in the expression of both the genes can be observed
with increase in number of days of differentiation.
10 (c) Markers for rods and cones (PR cells): Cone photoreceptor transcription
factor Thrβ, Rod photoreceptor specific transcription factor Nrl2 and photoreceptor
marker recoverin were analysed and the results have been depicted in Figure 3C.
It is apparent from the figure that all the cell markers display an increase in
expression with respect to increase in differentiation days.
15 (d) Pluripotency markers: Gene expression of pluripotency marker such as Oct4
and cell migration and division marker Blmp1 were also checked. As can be
observed in Figure 3D, both the genes displayed downregulation from day 7 day 77
of cell differentiation.
Figures 3E and 3F depict the quantification of PR cells based on the markers OTX2
20 and CRX, respectively.
[0095] Immunocytochemistry or Immunofluorescence study: For indirect
immunofluorescence, cells were harvested at appropriate time points and fixed in
2% paraformaldehyde for 20 minutes at room temperature (RT) followed by
washing three times in 1XPBS. Cells were then permeabilized for 5 minutes with
25 0.1% Triton X-100 (Sigma), then blocked for 1 hour at RT with 4% fetal bovine
serum (FBS), and incubated with primary antibodies (list of antibodies as mentioned
below in Table 6).
[0096] Followed by over-night primary antibody incubation at 48 hours, cells were
washed with 1PBS twice and probed with secondary antibodies for 1 hour at room
31
temperature After secondary antibody treatment cells were washed thoroughly with
1XPBS. For antibodies specific against membrane epitope cells were not
permeabilized. Cell nuclei were counterstained with DAPI (4’, 6-diamidino-2-
phenylindole; 100ng/ml) for 7 mins at RT and observed under fluorescent
microscope 5 (Olympus IX73).
[0097] Table 6 below depicts the list of antibodies used for the present study.
Table 6:
Vendor CATALOG NO. ANTIBODY NAME
Abcam ab80651 Mouse monoclonal MITF
Abcam ab13826 Mouse monoclonal RPE65
Abcam ab738 Mouse monoclonal Tyrosinase
Abcam ab4069 Mouse monoclonal Ezrin
Abcam ab109290 Rabbit monoclonal to SOX1
Abcam ab195045 Rabbit monoclonal PAX6
Santacruz sc271889 Mouse monoclonal Retinal RX
Abcam ab31928 Mouse monoclonal Recoverin
Abcam ab81213 Rabbit monoclonal BRN3A
Santacruz sc-377138 Mouse monoclonal CRX
Sigma T7451 Mouse monoclonal Acetylated Tubulin
Adipogen AG-20B-0020- C100 anti-Polyglutamylation Modification,
mAb
(GT335)
Abcam ab221391 Rabbit polyclonal NRL
Abcam ab53170 Rabbit polyclonal Thyroid Hormone
Abcam ab16288 M(ToHuRseB m) Ronecoecplotonral TRA-1-60
Abcam ab171380 Mouse monoclonal SOX-2
Abcam ab18976 Rabbit polyclonal Oct-4
Santacruz Sc-365519 Mouse monoclonal CHX10
Merck Millipore ABE1402 Rabbit polyclonal LHX2
Merck Millipore MAB5580 Mouse monoclonal anti-arrestin, visual
32
Abcam AB7260 Rabbit polyclonal GFAP
[0098] Figure 4 depicts phase contrast analysis and immunofluorescence studies of
differentiation of RPE from iPS cells. Figure 4A represents day 2 of the embryoid
bodies, and Figure 4A inset represents Oct4 staining in NcGMP1 iPSC, Figure 4B,
and 4C represents Day 20 rosette populations and Figure 4D represents 5 non-rosette
populations. Figure 4E and F represents pigmented RPE after rosette selection, and
Figures 4G-4I represent RPE like cells immunostained against Pax6, Nestin, Ezrin
and BRN3A antibodies, respectively. Figure 4J depicts Freeze thaw viability of RPE
progenitors, and Figure 4 K- 4L represents immunostaining against MITF and RX on
10 freeze thawed cells. Scale bars represent 100μm. Images unless mentioned are at 10X
magnification.
[0099] Figure 5 depicts characterization of mature RPE by immunofluorescence
analysis. Figures A and G represent purified mature RPE cells. Figures 5B and 5H
represent immunostaining of RPE for ZO-1. Figures 5C-5F represent
15 immunofluorescence images of transcription factors RX, CRX, MITF, and OTX2
respectively. Figure 5I represent the staining of phalloidin. Figures 5J-5L represent
immunostaining of mature RPE specific markers like Tyrosinase, RPE-65,
ARL13B, β-catenin (inset) in pigmented cells. Scale bars represent 100μm. Images
depicted unless mentioned are at 10X magnification. The depicted images clearly
20 represent the expression of appropriate markers and transcription factors expressed
by RPE. Hence, establishing the validity of the protocol as disclosed in the present
disclosure.
[00100] Figure 6A-6B depicts RPE quantification by flow cytometry of MITF and
RX in immature RPE respectively. Sandwich ELISA shows secretion of key growth
25 factors from fully mature and pigmented RPE compared to positive and negative
controls (Figure 6C). Figure 6 D represents Real time PCR based quantification of
key gene expression and it representation as heat map, respectively.
33
[00101] Figures 7A and 7B depict the phase contrast images of 3D retinal organoids
at day 60. The images show 3D self-organized retinal tissue formation in suspension
cultures.
[00102] Figures 7C and 7D depict the images post immunostaining of 3D
retinal organoids. 10μm section was taken and immunostained 5 for PR marker A)
OTX-2 and RPE marker B) MIT-F at day 25. Figure 7C depicts OTX-2 and RPE
marker and Figure 7D depicts the presence of MIT-F which represents the presence
of both RPE and PR in retinal organoid structures.
10 Advantages of the present disclosure:
[00103] Overall, the present disclosure provides with a protocol for producing
multiple cell types of retina from induced pluripotent stem cell. The protocol
described herein is a unified protocol that enables differentiation of iPSCs to retinal
cells- photoreceptors, retinal pigmented epithelial cells and 3D retinal organoids.
15 The single source of iPSC is grown under optimal condition of media with
inhibitors and inducers relevant for differentiation of different cell lineage
pertaining to the outer retina. Further, with the current protocol pigmentation
of RPEs is observed by 30-50 days, and the same is consistent and sustained
until 120 days and more. The protocol also does not require expensive and tedious
20 use of transwell plates. The protocol provides with retinal cell derivatives that have
better functional efficacy as well as consistent expression of the key markers
earlier in the course of differentiation.
34
I/We Claim:
1. A method for producing multiple cell types of retina from pluripotent stem
cells, said method comprising: (a) generating non-adherent suspension
embryoid bodies from the pluripotent stem cells in a medium 5 A, wherein the
medium A comprises growth factors, organic molecules, and lipid
concentrates; (b) culturing the non-adherent suspension embryoid bodies in a
medium B for 3-5 days, wherein the medium B comprises at least one WNT
pathway inhibitor and at least one ALK receptor inhibitor; (c) culturing the
10 embryoid bodies of step (b) on plates coated with at least one suitable
extracellular matrix in the medium B for 4-8 days; (d) culturing the embryoid
bodies of step (c) in a medium C for 5-20 days to form neural rosette like
structures and epithelial like retinal progenitor cells, wherein the medium C
does not comprise any inhibitor; (e) selecting the neural rosette like structures
15 using a selection reagent, wherein the selection reagent allows preferential
isolation and enrichment of the neural rosette like structures; (f) culturing
the neural rosette like structures of step (e) on plates coated with at least
one suitable extracellular matrix in a modified medium C until confluence for
formation of photoreceptor progenitors, wherein the modified medium C
20 comprises at least one ROCK inhibitor and NOTCH inhibitor; (g) culturing
the photoreceptor progenitors in the modified medium C for 8-12 weeks
to form mature photoreceptor cells; (h) selecting and culturing the epithelial
like retinal progenitor cells obtained in step (d) in the medium C for 5-7 days
for formation of retinal pigmented epithelial progenitors; (i) culturing the
25 retinal pigmented epithelial progenitors of step (h) in a medium D for 15-30
days to obtain mature retinal pigmented epithelial cells, wherein the medium D
comprises taurine, hydrocortisone and triido-thyronine;
35
(j) culturing the non-adherent suspension embryoid bodies of step (a) in the
medium B for 8-10 days; and (k) culturing the non-adherent suspension
embryoid bodies of step (j) in the medium C for 8-12 weeks for formation of
3D retinal organoids, wherein the method is a unified method for producing
multiple cell 5 types of retina.
2. A method for producing multiple cell types of retina from pluripotent stem
cells, said method comprising: (a) o b t a i n i n g a d h e r e n t c u l t u r e s b y
direct differentiation from the pluripotent stemcells in a medium A, wherein
10 the medium A comprises growth factors, organic molecules, and lipid
concentrates; (b) culturing the adherent cultures in a medium B for 3-5 days,
wherein the medium B comprises at least one WNT pathway inhibitor and
at least one ALK receptor inhibitor; (c) culturing the adherent cultures
of step (b) on plates coated with at least one suitable extracellular matrix in
15 the medium B for 4-8 days; (d) culturing the adherent cultures of step (c)
in a medium C for 5-20 days to form neural rosette like structures and
epithelial like retinal progenitor cells, wherein the medium C does not
comprise any inhibitor; (e) selecting the neural rosette like structures using a
selection reagent, wherein the selection reagent allows preferential isolation
20 and enrichment of the neural rosette like structures; (f) culturing the neural
rosette like structures of step (e) on plates coated with at least one suitable
extracellular matrix in a modified medium C until confluence for formation of
photoreceptor progenitors, wherein the modified medium C comprises at least
one ROCK inhibitor and NOTCH inhibitor; (g) culturing the photoreceptor
25 progenitors in the modified medium C for 8-12 weeks to form mature
photoreceptor cells; (h) selecting and culturing the epithelial like retinal
progenitor cells obtained in step (d) in the medium C for 5-7 days for formation
of retinal pigmented epithelial progenitors; (i) culturing the retinal pigmented
epithelial progenitors of step (h) in a medium D for 15-45 days to obtain
36
mature retinal pigmented epithelial cells, wherein the medium D comprises
taurine, hydrocortisone and triido-thyronine, to obtain multiple cell types of
retina from pluripotent stem cells.
3. The method as claimed in claim 1, wherein the multiple cell 5 types of retina
comprise photoreceptor cells, retinal pigmented epithelial cells and 3D retinal
organoids.
4. The method as claimed in the claim 2, wherein the multiple cell types of
10 retina comprise photoreceptor cells, and retinal pigmented epithelial cells.
20
5. The method as claimed in claim 1, wherein generating the non-adherent
suspension embryoid bodies from the pluripotent stem cell comprises:
(a) growing the pluripotent stem cells on a plate coated with at least one
15 suitable extracellular matrix in a medium A until confluence, wherein the
medium A comprises growth factors, organic molecules and lipid concentrates;
25 (b) dislodging the pluripotent stem cells of step (a) using a cell detachment
enzyme solution to obtain detached pluripotent stem cells;
(c) neutralizing the detached pluripotent stem cells with a suitable
20 neutralization medium to obtain neutralized pluripotent stem cell, wherein the
neutralization medium comprises 5 -20% KOSR; and
(d) seeding the neutralized pluripotent stem cells on a non-adherent suspension
culture plate in a modified medium A for generating the non-adherent
suspension embryoid bodies, wherein the modified medium A comprises at
25 least one ROCK inhibitor and NOTCH inhibitor.
6. The method as claimed in any of the claims 1 or 2, wherein the mature retinal
pigmented epithelial cells obtained in step (i) is further passaged, comprising:
37
(a) incubating the retinal pigmented epithelial cells with 0.05% - 0.5% trypsin
EDTA solution for detachment of non-pigmented and non-retinal pigmented
epithelial cells for 2-10 minutes;
10 (b) discarding the non-pigmented and non-retinal pigmented epithelial cells to
obtain a pure population of retinal pigmented 5 epithelial cells;
(c) incubating the pure population of retinal pigmented epithelial cells with
medium E, wherein the medium E comprises accutase enzyme and 0.2-0.5%
trypsin EDTA;
(d) neutralizing the pure population of retinal pigmented epithelial cells of
10 step (c) using neutralization medium comprising 5-20% KOSR, to obtain
neutralized pure population of retinal pigmented epithelial cells; and
(e) culturing the neutralized pure population of retinal pigmented epithelial cells
on a plate coated with at least one suitable extracellular matrix in the medium
D.
15
7. The method as claimed in claim 1, wherein the non-adherent suspension
embryoid bodies generated in step (d) is further maintained in the modified
medium A for 24-48 hours.
20 8. The method as claimed in claim 2, wherein the adherent cultures generated
in step (d) is further maintained in the modified medium A for 24-48 hours.
9. The method as claimed in any of the claims 1 or 2, wherein the at least
one WNT pathway inhibitor is selected from a group consisting of 4-
25 (1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7- methano-2H-isoindol-2-yl)-N-8-
quinolinyl-Benzamide, 5-(Phenylsulfonyl)-N- piperidin-4-yl-2-
(trifluoromethyl) benzene sulfonamide, 2-(2',3-Dimethyl-[2,4'-bipyridin]-
5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl) acetamide, 2-(4-(2-methylpyridin-4-
38
yl)phenyl)-N-(4-(pyridin-3-yl)phenyl) acetamide, 8-Tetrahydro-2-[4-
(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one, and
combinations thereof.
10. The method as claimed in any of the claims 1 or 2, wherein 5 the at least one
ALK receptor inhibitor is selected from a group consisting of 4-[4-(1,3-
benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl] benzamide, 4-(6-(4-
(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, 3-[(1R)-1-
(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-
10 2-amine, 5-chloro-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-
yl]phenyl]-4-N-(2-propan-2-ylsulfonylphenyl)pyrimidine-2,4-diamine, and 9-
ethyl-6,6-dimethyl-8-(4-morpholin-4-ylpiperidin-1-yl)-11-oxo-5Hbenzo[
b]carbazole-3-carbonitrile, 5-chloro-2-N-(5-methyl-4-piperidin-4-yl-2-
propan-2-yloxyphenyl)-4-N-(2-propan-2-ylsulfonylphenyl) pyrimidine-2,4-
15 diamine, and combinations thereof
11. The method as claimed in any one of the claims 1 or 2, wherein the at least
one suitable extracellular matrix is selected from the group consisting of
laminin, vitronectin, fibronectin, collagen, poly-L-lysine, poly-L-ornithine, and
20 combinations thereof.
12. The method as claimed in any one of the claims 1 or 2, wherein the
medium C further comprises DMEM/F12, knock out serum replacement, and
non-essential amino acids.
25
13. The method as claimed in any one of the claims 1 or 2, wherein the at
least one ROCK1 inhibitor is selected from a group consisting of 4-[(1R)-1-
aminoethyl]-N- pyridin-4-ylcyclohexane-1-carboxamide, 4-[(1R)-1-
39
aminoethyl]-N-(1H-pyrrolo[2,3- b] pyridin-4-yl)benzamide, 6-
aminochromen-4-one, 5-(1,4-diazepan-1-
ylsulfonyl)isoquinoline, 4-methyl-5-[[(2S)-2-methyl-1,4-
diazepan-1- yl]sulfonyl]isoquinoline, and combinations thereof.
5
14. The method as claimed in any one of the claims 1 or 2, wherein the at
least one NOTCH inhibitor is selected from a group consisting of N-[N-(3,5-
Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), N-[cis-
4-[(4-Chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-
trifluoromethanesulfonamide, 2-[(1R)-1-[[(4-Chlorophenyl)10 sulfonyl](2,5-
difluorophenyl)amino]ethyl-5-fluorobenzenebutanoic acid, 5-Chloro-N-[(1S)-
3,3,3-trifluoro-1-(hydroxymethyl)-2-(trifluoromethyl)propyl]-2-
thiophenesulfonamide, N-[(1S)-2-[[(7S)-6,7-Dihydro-5-methyl-6-oxo-5Hdibenz[
b,d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-
15 difluorobenzeneacetamide, (R)-2-Fluoro-α-methyl[1,1'-biphenyl]-4-acetic
acid, N-[(1S)-2-[[(3S)-2,3-Dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-
benzodiazepin-3-yl]amino]-1-methyl-2-oxoethyl]-3,5-
difluorobenzeneacetamide, 3,5-Bis(4-nitrophenoxy)benzoic acid, (5S)-(tert-
Butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanoyl)-L20
leucy-L-phenylalaninamide, 7-Amino-4-chloro-3-methoxy-1H-2-benzopyran,
(2S)-N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-
dimethylethyl ester, and combinations thereof.
15. The photoreceptor cells produced by the method as claimed in any one of
25 the claims 1 or 2.
16. The retinal pigmented epithelial cells produced by method as claimed in
any one of the claims 1 or 2.
40
17. The 3D retinal organoids produced by method as claimed in claim 1.
18. A pharmaceutical composition comprising the photoreceptor cells as
claimed 5 in claim 15.
19. A pharmaceutical composition comprising the retinal pigmented epithelial
cells as claimed in claim 16.
10 20. A pharmaceutical composition comprising the 3D retinal organoids as
claimed in claim 17.
21. The composition as claimed in any one of the claims 18-20 for use in
manufacture of a medicament.
15
22. A medicament comprising the composition as claimed in claim 21, for
use in treatment of retinal degenerative diseases.
23. The medicament as claimed in claim 22, wherein the retinal regenerative
20 diseases is selected from the group consisting of Retinitis Pigmentosa, Best
disease, Stargardt disease, Usher syndrome, rod-cone dystrophy, and age
related macular degeneration.