Method for producing retinal pigment epithelial cells
Field of the invention
The invention relates to methods for producing retinal pigment epithelial (RPE) cells fr5 om
pluripotent cells. The invention also relates to the cells obtained or obtainable by such
methods as well as to their use for the treatment of retinal diseases. The invention also
relates to a process for expanding RPE cells.
10 Background of the invention
The retinal pigment epithelium is the pigmented cell layer outside the neurosensory retina
between the underlying choroid (the layer of blood vessels behind the retina) and overlying
retinal visual cells (e.g., photoreceptors rods and cones). The retinal pigment epithelium is
15 critical to the function and health of photoreceptors and the retina. The retinal pigment
epithelium maintains photoreceptor function by recycling photopigments, delivering,
metabolizing, and storing vitamin A, phagocytosing rod photoreceptor outer segments,
transporting iron and small molecules between the retina and choroid, maintaining Bruch's
membrane and absorbing stray light to allow better image resolution. Degeneration of the
20 retinal pigment epithelium can cause retinal detachment, retinal dysplasia, or retinal atrophy
that is associated with a number of vision-altering ailments that result in photoreceptor
damage and blindness, such as, choroideremia, diabetic retinopathy, macular degeneration
(including age-related macular degeneration), retinitis pigmentosa, and Stargardt's Disease.
25 A potential treatment for such diseases is the transplantation of RPE cells into the retina of
those affected with the diseases. It is believed that replenishment of retinal pigment epithelial
cells by their transplantation may delay, halt or reverse degeneration, improve retinal function
and prevent blindness stemming from such conditions. It has been demonstrated in animal
models that photoreceptor rescue and preservation of visual function could be achieved by
30 subretinal transplantation of RPE cells (see for example Coffey, PJ et al. Nat. Neurosci.
2002:5, 53-56; Sauve, Yet al. Neuroscience 2002: 114, 389-401). Therefore, there is a high
interest in finding ways to produce RPE cells, for example from pluripotent cells, as a source
for cell transplantation for the treatment of retinal diseases.
35 The potential of mouse and non-human primate embryonic stem cells to differentiate into
RPE cells, and to survive and attenuate retinal degeneration after transplantation, has been
demonstrated. Spontaneous differentiation of human embryonic stem cells into RPE cells
2
was shown (see for example WO2005/070011). However, the efficiency and reproducibility
of such process was low. Therefore, there is a need for methods for producing RPE cells
which are well controlled, reproducible, efficient and/or suitable for scale up and for
producing RPE cells for drug screening, disease modeling and/or therapeutic use.
5
Summary of the invention
The present invention relates to methods for producing RPE cells. It is demonstrated that the
methods provide robust and reproducible differentiation of pluripotent cells such as human
10 embryonic stem cells (hESCs) to give rise to RPE cells. In addition, the methods provided
herein are easily scalable to give a high yield of RPE cells. Methods disclosed herein can be
used, for example without limitation, for reproducibly and efficiently differentiating pluripotent
cells such as hESC into RPE cells in xeno-free conditions.
15 Methods for producing RPE cells are provided herein. In some embodiments, the method
comprises the steps of:
(a) culturing pluripotent cells in the presence of a first SMAD inhibitor and a second SMAD
inhibitor;
(b) culturing the cells of step (a) in the presence of a Bone Morphogenetic Protein (BMP)
20 pathway activator and in the absence of the first and second SMAD inhibitors; and,
(c) replating the cells of step (b).
In some embodiments of said method, the method further comprises the following steps:
(d) culturing the replated cells of step (c) in the presence of an activin pathway activator;
(e) replating the cells of step (d); and,
25 (f) culturing the replated cells of step (e).
In another embodiment of said method,
step (b) further comprises, after culturing the cells in the presence of the BMP pathway
activator, culturing the cells for at least 10 days in the absence of the BMP pathway activator;
step (c) comprises replating the cells of step (b) having a cobblestone morphology; and said
30 method further comprising the step of:
(d) culturing the replated cells of step (c).
Also provided are methods for expanding RPE cells. In some embodiments, the method
comprises the following steps:
(a) plating RPE cells at a density between 1000 and 100000 cells/cm2, and,
35 (b) culturing said RPE cells in the presence of SMAD inhibitor, cAMP or an agent which
increases the intracellular concentration of cAMP.
3
Also provided are methods for purifying RPE cells comprising:
a) providing a cell population comprising RPE cells and non RPE cells;
b) increasing the percentage of RPE cells in the cell population by enriching the cell
population for cells expressing CD59.
5
Also provided are RPE cells obtained or obtainable by a method disclosed herein.
Also provided are pharmaceutical compositions. The pharmaceutical compositions comprise
RPE cells suitable for transplantation into the eye of a subject affected with a retinal disease.
10 In some embodiments, the pharmaceutical composition comprises a structure suitable for
supporting RPE cells. In some embodiments, the pharmaceutical composition comprises a
porous membrane and RPE cells. In some embodiments, the pores of the membrane are
between about 0.2μm and about 0.5μm in diameter and the pore density are between about
1x107 and about 3x108 pores per cm2. In some embodiments, the membrane is coated on
15 one side with a coating supporting RPE cells. In some embodiments, the coating comprises
a glycoprotein, preferably selected from laminin or vitronectin. In some embodiments, the
coating comprises vitronectin. In some embodiments, the membrane is made of polyester.
Also provided are methods for the treatment of a retinal disease in a subject. In some
20 embodiments, the method comprises administering RPE cells of the present invention to a
subject affected by or at risk for retinal disease, thereby treating the retinal disease.
Brief description of drawings
25 Figure 1A shows a schematic representation of a specific example of the early and late
replating methods.
Figures 1B and 1C show graphs indicating the percentage of cells expressing PAX6 and
OCT4 as measured by immunocytochemistry at different time points during treatment with
SMAD inhibitors. Figure 1B: samples induced with LDN/SB. Figure 1C: samples not induced
30 with LDN/SB.
Figure 1D shows graphs indicating the percentage of cells expressing PAX6 (top graph) and
OCT4 (Bottom graph) as measured by immunocytochemistry after 2 days (LDN/SB 2D) or 5
days (Control+) treatment with SMAD inhibitors.
Figure 2A shows graphs indicating the relative expression of Mitf (top graph) and Silv
35 (PMEL17) (bottom graph) as measured by qPCR under different conditions. Figure 2B shows
graphs indicating the percentage of cells expressing MITF (top graph) and PMEL17 (bottom
graph) as measured by immunocytochemistry. Figures 2A and 2B show that treatment with a
4
BMP pathway activator after step (a) is essential to induce the expression of MITF and
PMEL17.
Figure 3 shows graphs indicating the percentage of cells expressing MITF as measured by
immunocytochemistry (top graph) or qPCR (bottom graph) after treatment with different BMP
pathway activators. Figure 3 shows that different BMP pathway activators can be used i5 n
step (b) of the method disclosed herein.
Figure 4A shows graphs indicating the percentage of cells expressing CRALBP as measured
by immunocytochemistry under different conditions.
Figure 4B shows a graph indicating the percentage of cells expressing MERTK as measured
10 by immunocytochemistry under different conditions.
Figure 4C shows graphs indicating the relative expression of Rlbp1 (CRALBP) (top graph)
and Mitf (bottom graph) as measured by qPCR under different conditions.
Figure 4D shows graphs indicating the relative expression of Mertk (top graph) and Best1
(bottom graph) as measured by qPCR under different conditions.
15 Figure 4E shows graphs indicating the relative expression of Silv (PMEL17) (top graph) and
Tyr (bottom graph) as measured by qPCR under different conditions.
Figure 5 shows a graph indicating the percentage of cells expressing CRALBP at D9-19 as
measured by immunocytochemistry under different conditions. Figure 5 shows that activin A
is a suitable activin pathway activator for use in the method disclosed herein and that a short
20 exposure to activin A is sufficient to induce expression of RPE markers.
Figures 6 and 7 show graphs indicating the percentage of cells expressing PMEL17 (top
graph) and CRALBP (bottom graph) at D9-19-20 in 96 well plates (Fig.6) and 384 well plates
(Fig.7) as measured by immunocytochemistry when cells are replated (step (e) of the early
replate embodiment) at different seeding densities on different plates and cultured in media
25 optionally comprising cAMP. Figures 6 and 7 show inter alia that different seeding densities
can be used in step (e).
Figure 8A shows the cells at Day 49 (step (b)) of the late replate embodiment after treatment
with SMAD inhibitors, BMP pathway activator and culture in basic medium until Day 49.
Figure 8B shows the cells after 12 days of culture (step (d)) post replating. Figure 8C shows
30 graphs indicating the percentage of cells expressing PMEL17 (top graph) and CRALBP
(bottom graph) as measured by immunocytochemistry after 15 days of culture post replating.
Figure 9A shows a Principal Component Analysis (PCA) plot of 7 RPE samples generated by
directed differentiation along with RPE cells generated by spontaneous differentiation as well
as de-differentiated controls. Figure 9B shows the loading plots used for PCA which indicates
35 contribution of each of the genes tested to the clustering of the samples. Figure 9C shows
the comparison of whole genome transcript profiling of RPE cells obtained by Directed
5
Differentiation (both Early and Late replating as disclosed in examples 1 and 8), RPE cells
obtained by Spontaneous Differentiation and hES cells.
Figure 10A shows a graph indicating the ratio of concentration of VEGF to concentration of
PEDF in the spent media of the bottom and top chambers of the Transwell® at week 10.
Figure 10A is consistent with the conclusion that the cells obtained by the method of th5 e
invention are RPE cells.
Figure 10B shows a graph depicting the increase of PEDF and VEGF in the spent media of
cells cultured after the replating step (c). Figure 10B is consistent with the conclusion that the
cells obtained by the method of the invention are RPE cells.
10 Figure 11A is a schematic representation of the Epithelial-Mesenchymal Transition and
Mesenchymal-Epithelial Transition occurring during RPE cells expansion.
Figure 11B shows a graph indicating the number of cells (Hoescht positive nuclei per frame
imaged) obtained after expansion of RPE cells under different conditions. Figure 11B shows
that the use of cAMP or an agent which increases the intracellular concentration of cAMP
15 step increases the yield of the expansion step.
Figure 11C shows a graph indicating the percentage of cells expressing PMEL17 as
measured by immunocytochemistry after expansion of RPE cells optionally in the presence
of cAMP.
Figure 11D shows a graph indicating the percentage of cells expressing PMEL17 as
20 measured by immunocytochemistry after expansion of RPE cells optionally in the presence
of an agent that increases intracellular cAMP such as Forskolin.
Figure 11E shows a graph indicating the percentage of EdU incorporation in RPE cells
expanded in the presence of cAMP.
Figure 11F shows a graph indicating the number of cells per cm2 obtained after expansion of
25 RPE cells in the presence of cAMP.
Figure 11G shows a graph indicating the percentage of cells expressing Ki67 at D14 as
measured by immunocytochemistry after expansion of RPE cells optionally in the presence
cAMP.
Figure 11H shows a graph indicating the percentage of cells expressing PMEL17 at D14 as
30 measured by immunocytochemistry after expansion of RPE cells optionally in the presence
cAMP.
Figure 11I shows a graph indicating the expression of Mitf at week 5 as measured by qPCR
after expansion of RPE cells optionally in the presence cAMP.
Figure 11J shows a graph indicating the expression of Silv at week 5 as measured by qPCR
35 after expansion of RPE cells optionally in the presence cAMP.
Figure 11K shows a graph indicating the expression of Tyr at week 5 as measured by qPCR
after expansion of RPE cells optionally in the presence cAMP.
6
Figure 12A shows a graph indicating the percentage of EdU incorporation in RPE cells
expanded in the presence of a SMAD inhibitor.
Figure 12B shows a graph indicating the expression of Best1 at week 5 as measured by
qPCR after expansion of RPE cells optionally in the presence of a SMAD inhibitor.
Figure 12C shows a graph indicating the expression of Rlbp1 at week 5 as measured 5 sured by
qPCR after expansion of RPE cells optionally in the presence of a SMAD inhibitor.
Figure 12D shows a graph indicating the expression of Grem1 at week 5 as measured by
qPCR after expansion of RPE cells optionally in the presence of a SMAD inhibitor.
Figure 13A shows a graph indicating the percentage of EdU incorporation at Day 14 in RPE
10 cells expanded in the presence of an antibody against TGF1 and TGF2 ligands.
Figure 13B shows a graph indicating the percentage of cells expressing PMEL17 at D14 as
measured by immunocytochemistry after expansion of RPE cells optionally in the presence
of an antibody against TGF1 and TGF2 ligands.
Figure 13C, 13D, 13E, 13F, 13G and 13H show respectively a graph indicating the
15 percentage of cells expressing Best1, Merkt, Grem1, Silv, Lrat and Rpe65 as measured by
qPCR after expansion of RPE cells optionally in the presence of an antibody against TGF1
and TGF2 ligands.
Figure 14A shows a graph indicating the relative expression of hESC markers as measured
by qPCR in cells stained with an anti-CD59 antibody triaged by flow cytometry.
20 Figure 14B shows a graph indicating the relative expression of RPE markers as measured by
qPCR in cells stained with an anti-CD59 antibody triaged by flow cytometry.
Figure 15A, 15B, 15C and 15D show respectively the percentage of cells expressing OCT4,
LHX2, PAX6 and CRALBP at D2, D9 (and D9-19 for CRALBP) as measured by
immunocytochemistry during the differentiation of iPSC in RPE cells.
25 Figure 15E, 15F and 15G show respectively the percentage of cells expressing Best1, Mertk
and Silv as measured by qPCR after second replating (D9-19-45) in a directed differentiation
protocol using iPSC as starting material. ESDD means RPE cells obtained by directed
differentiation using hESC as starting material. IPSDD means RPE cells obtained by directed
differentiation using iPSC as starting material.
30
Detailed description
In some embodiments, the term “pluripotent cell” refers to a cell capable of differentiating to
cell types of the three germ layers (e.g., can differentiate to ectodermal, mesodermal and
35 endodermal cell types) under the appropriate conditions. Pluripotent cells can also be
maintained in culture in vitro for a prolonged period of time in an undifferentiated state. In a
7
preferred embodiment, the pluripotent cells are of vertebrate, in particular mammalian,
preferably human, primate or rodent origin. Preferred pluripotent cells are human pluripotent
cells. Examples of pluripotent cells are embryonic stem cells or induced pluripotent stem
cells. In some embodiments, the pluripotent cells are obtained by a method which does not
involve destruction of human embry5 os.
In some embodiments, the pluripotent cell is an embryonic stem cell (ESC).
In some embodiments, ESC refers to stem cells derived from an embryo. In some
10 embodiments, the embryo is obtained from in vitro fertilized embryos.
In some embodiments, ESC refers to cells derived from the inner cell mass of blastocysts or
morulae that have been serially passaged as cell lines. In some embodiments, said
blastocysts are obtained from an in vitro fertilized embryo. In some embodiments, said
15 blastocysts are obtained from a non-fertilized oocyte which is parthenogenetically activated
to cleave and develop to the blastocyst stage.
ESC may be obtained by methods known to the skilled person (see for example US5843780,
which is herein incorporated by reference in its entirety).
20
For example, for the isolation of hESCs from a blastocyst, the zona pellucida is removed and
the inner cell mass is isolated by immunosurgery, in which the trophectoderm cells are lysed
and removed from the intact inner cell mass by gentle pipetting. The inner cell mass is then
plated in a tissue culture flask containing the appropriate medium which enables its
25 outgrowth. Following 9 to 15 days, the inner cell mass derived outgrowth is dissociated into
clumps either by mechanical dissociation or by enzymatic digestion and the cells are then replated
on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology
are individually selected by micropipette, mechanically dissociated into clumps, and replated.
Resulting ESCs are then routinely split every 1-2 weeks.
30
In some embodiments, the term ESC refers to cells isolated from one or more blastomeres of
an embryo, preferably without destroying the remainder of the embryo (see, for example
US20060206953 or US20080057041, which are herein incorporated by reference in their
entirety).
35
In a preferred embodiment, the pluripotent cell is a human embryonic stem cell. In a
preferred embodiment, the pluripotent cell is a human embryonic stem cell obtained without
8
destruction of an embryo. In a preferred embodiment, the pluripotent cell is a human
embryonic stem cell originating from a well established cell line such as MA01, MA09, ACT-
4, H1, H7, H9, H14, WA25, WA26, WA27, Shef-1, Shef-2, Shef-3, Shef-4 or ACT30
embryonic stem cell.
5
In some embodiments, ESC, regardless of their source or the particular method used to
produce them, can be identified based on the: (i) ability to differentiate into cells of all three
germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and (iii) ability to
produce teratomas when transplanted into immunocompromised animals.
10
In some embodiments, the pluripotent cell is an induced pluripotent stem cell (iPSC).
In some embodiments, an iPSC is a pluripotent cell derived from a non pluripotent cell such
as for example an adult somatic cell, by reprogramming said somatic cell for example by
15 expressing or inducing expression of a combination of factors. IPSCs are commercially
available or can be obtained by methods known to the skilled person. IPSCs can be
generated using for example fetal, postnatal, newborn, juvenile, or adult somatic cells. In
certain embodiments, factors that can be used to reprogram somatic cells to pluripotent stem
cells include, for example, a combination of Oct4 (sometimes referred to as Oct 3/4), Sox2,
20 c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells
to pluripotent stem cells include, for example, a combination of Oct-4, Sox2, Nanog, and
Lin28 (see for example EP2137296, which is herein incorporated by reference in its entirety).
In some embodiments, the iPSCs are obtained by reprogramming a somatic cell using a
combination of small molecule compounds (see for example, Science, Vol. 341 no.6146,
25 pp.651-654, which is herein incorporated by reference in its entirety).
In a preferred embodiment, the pluripotent cell is a human induced pluripotent stem cell. In a
preferred embodiment, the pluripotent cell is an induced pluripotent stem cell derived from a
human adult somatic cell.
30
IPSC can be obtained for example using methods disclosed in US20090068742,
US20090047263, US20090227032, US20100062533, US20130059386, WO2008118820, or
WO2009006930, which are herein incorporated by reference in their entirety.
35 In some embodiments, the term “SMAD inhibitor” refers to an inhibitor of Small Mothers
Against Decapentaplegic (SMAD) protein signaling.
9
In some embodiments, the term “first SMAD inhibitor” refers to an inhibitor of BMP type 1
receptor ALK2. In some embodiments, the first SMAD inhibitor is an inhibitor of BMP type 1
receptors ALK2 and ALK3. In some embodiments, the first SMAD inhibitor prevents Smad1,
Smad5 and/or Smad8 phosphorylation. In some embodiments, the first SMAD inhibitor is a
dorsomorphin derivative. In some embodiments, the first SMAD inhibitor is selected fro5 m
dorsomorphin, noggin, chordin or 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-
yl)quinoline (LDN193189). In a preferred embodiment, the first SMAD inhibitor is 4-(6-(4-
(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (LDN193189) or a salt or
hydrate thereof.
10
LDN193189 is a commercially available compound of formula
N
HN
N
N
N
N
.
15 In some embodiments, the term “second SMAD inhibitor” refers to an inhibitor of
transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors.
In some embodiments, the second SMAD inhibitor is an inhibitor of ALK5. In some
embodiments, the second SMAD inhibitor is an inhibitor of ALK5 and ALK4. In some
embodiments, the second SMAD inhibitor is an inhibitor of ALK5 and ALK4 and ALK7. In
20 some embodiments, the second SMAD inhibitor is 4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-
2-yl)-1H-imidazol-2-yl)benzamide (SB-431542) or a salt or hydrate thereof.
SB-431542 is a commercially available compound of formula
10
In some embodiments, the second SMAD inhibitor is selected from :
4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide;
2-methyl-5-(6-(m-tolyl)-1H-imidazo[1,2-a]imidazol-5-yl)-2H-benzo[d][1,2,3]triazol5 e;
2-(6-methylpyridin-2-yl)-N-(pyridin-4-yl)quinazolin-4-amine;
2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine;
4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)phenol;
2-(4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[3,2-c]pyridine;
10 4-(5-(3,4-dihydroxyphenyl)-1-(2-hydroxyphenyl)-1H-pyrazol-3-yl)benzamide;
2-(5-chloro-2-fluorophenyl)-N-(pyridin-4-yl)pteridin-4-amine; or,
6-methyl-2-phenylthieno[2,3-d]pyrimidin-4(3H)-one;
or a salt or hydrate thereof.
The above compounds are commercially available or can be prepared by processes known
15 to the skilled person (see for example Surmacz et Al, Stem Cells 2012;30:1875-1884).
In some embodiments, the second SMAD inhibitor is selected from 3-(6-Methyl-2-pyridinyl)-
N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A 83-01), 2-(5-Benzo[1,3]dioxol-5-
yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine (SB-505124), 7-(2-morpholinoethoxy)-4-(2-
20 (pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline (LY2109761) or 4-[3-(2-
pyridinyl)-1H-pyrazol-4-yl]-quinoline (LY364947).
In some embodiments, the BMP pathway activator comprises a BMP. In some embodiments,
the BMP pathway activator comprises a BMP selected from BMP2, BMP3, BMP4, BMP6,
25 BMP7, BMP8, BMP9, BMP10, BMP11 or BMP15. In some embodiments, the BMP pathway
activator is a BMP homodimer, preferably a BMP2, BMP3, BMP4, BMP6, BMP7, BMP8,
BMP9, BMP10, BMP11 or BMP15 homodimer. In some embodiments, the BMP pathway
activator is a BMP homodimer, preferably a BMP2, BMP3, BMP4, BMP6, BMP7, or BMP8
homodimer. In some embodiments, the BMP pathway activator is a BMP heterodimer,
30 preferably comprising a BMP selected from BMP2, BMP3, BMP4, BMP6, BMP7, BMP8,
11
BMP9, BMP10, BMP11 or BMP15. In some embodiments, the BMP pathway activator is a
BMP heterodimer, preferably comprising a BMP selected from BMP2, BMP3, BMP4, BMP6,
BMP7 or BMP8. In some embodiments, the BMP pathway activator is a BMP2/6
heterodimer, a BMP4/7 heterodimer or a BMP3/8 heterodimer. In some embodiments, the
BMP pathway activator is a BMP4/7 heterodimer5 .
In some embodiments, the BMP pathway activator is a small molecule activator of BMP
signaling (see for example PLOS ONE, March 2013, Vol.8 (3), e59045, which is herein
incorporated by reference in its entirety).
10 In some embodiments, the term “Retinal Pigment Epithelial cell” or “RPE cell” refers to a cell
having the morphological and functional attributes of an adult RPE cell, preferably an adult
human RPE cell.
In some embodiments, the RPE cell has the morphological attributes of an adult RPE cell
15 preferably an adult human RPE cell. In some embodiments, the RPE cell has a cobblestone
morphology. In some embodiments, the RPE cell is pigmented. The shape, morphology and
pigmentation of RPE cells can be observed visually.
In some embodiments, the RPE cell expresses at least one of the following RPE markers:
20 MITF, PMEL17, CRALBP, MERTK, BEST1 and ZO-1. In some embodiments, the RPE cell
expresses at least two, three, four or five of the following RPE markers: MITF, PMEL17,
CRALBP, MERTK, BEST1 and ZO-1. In some embodiments, the expression of the RPE
markers is measured by immunocytochemistry. In some embodiments, the expression of the
RPE markers is measured by immunocytochemistry as detailed in the example section. In
25 some embodiments, the expression of markers is measured by quantitative PCR. In some
embodiments, the expression of the RPE markers is measured by quantitative PCR as
detailed in the example section.
In some embodiments, the RPE cell does not express Oct4
30
In some embodiments, the RPE cell has the functional attributes of an adult RPE cell,
preferably an adult human RPE cell. In some embodiments, the RPE cell secretes VEGF. In
some embodiments, the RPE cell secretes PEDF. In some embodiments, the RPE cell
secretes PEDF and VEGF. In some embodiments, VEGF and/or PEDF secretion by RPE
35 cells is measured by a quantitative immunoassay. In some embodiments, VEGF and/or
PEDF secretion by RPE cells is measured as disclosed in the examples.
12
In a preferred embodiment, the RPE cell has a cobblestone morphology, is pigmented and
expresses at least one of MITF, PMEL17, CRALBP, MERTK, BEST1 and ZO-1. In a
preferred embodiment, the RPE cell has a cobblestone morphology, is pigmented and
expresses at least two of MITF, PMEL17, CRALBP, MERTK, BEST1 and ZO-1. In a
preferred embodiment, the RPE cell has cobblestone morphology, is pigmented, ex5 presses
at least two of MITF, PMEL17, CRALBP, MERTK, BEST1 and ZO-1 and secretes VEGF and
PEDF.
When a parameter is defined as “between a low value and high value”, such low and high
10 value should be considered as part of the defined range.
Early Replating
In one embodiment (early replating embodiment), the invention relates to a method for
15 producing RPE cells comprising the following steps:
(a) culturing pluripotent cells in the presence of a first SMAD inhibitor and a second SMAD
inhibitor;
(b) culturing the cells of step (a) in the presence of a BMP pathway activator and in the
absence of the first and second SMAD inhibitors; and,
20 (c) replating the cells of step (b).
In some embodiments, in step (a), the pluripotent cells are cultured for at least 1 day. In
some embodiments, in step (a), the pluripotent cells are cultured for at least 1 day, at least 2
days, at least 3 days or at least 4 days. In some embodiments, in step (a), the pluripotent
25 cells are cultured for between 2 and 10 days. In some embodiments, in step (a), the
pluripotent cells are cultured for between 2 and 6 days. In some embodiments, in step (a),
the pluripotent cells are cultured for between 3 and 5 days. In some embodiments, in step
(a), the pluripotent cells are cultured for about 4 days.
30 In some embodiments, in step (a), the concentration of first SMAD inhibitor is between 0.5nM
and 10μM. In some embodiments, in step (a), the concentration of first SMAD inhibitor is
between 1nM and 5μM. In some embodiments, in step (a), the concentration of first SMAD
inhibitor is between 1nM and 2μM. In some embodiments, in step (a), the concentration of
first SMAD inhibitor is between 500nM and 2μM. In some embodiments, in step (a), the
35 concentration of first SMAD inhibitor is about 1μM. In a preferred embodiment, the first
SMAD inhibitor is LDN193189.
13
In some embodiments, in step (a), the concentration of second SMAD inhibitor is between
0.5nM and 100μM. In some embodiments, in step (a), the concentration of second SMAD
inhibitor is between 100nM and 50μM. In some embodiments, in step (a), the concentration
of second SMAD inhibitor is between 1μM and 50μM. In some embodiments, in step (a), the
concentration of second SMAD inhibitor is between 5μM and 20μM. In some embodiments5 ,
in step (a), the concentration of second SMAD inhibitor is at least 5μM. In some
embodiments, in step (a), the concentration of second SMAD inhibitor is about 10μM. In a
preferred embodiment, the second SMAD inhibitor is SB-431542.
10 In some embodiments, in step (b), the concentration of BMP pathway activator is between
1ng/mL and 10μg/mL. In some embodiments, in step (b), the concentration of BMP pathway
activator is between 5ng/mL and 1μg/mL. In some embodiments, in step (b), the
concentration of BMP pathway activator is between 50 ng/mL and 500ng/mL. In some
embodiments, in step (b), the concentration of BMP pathway activator is about 100ng/mL. In
15 a preferred embodiment the BMP pathway activator is a BMP4/7 heterodimer.
In some embodiments, in step (b), the cells are cultured for at least 1 day. In some
embodiments, in step (b), the cells are cultured for at least 1 day, at least 2 days, at least 3
days or at least 4 days. In some embodiments, in step (b), the cells are cultured for at least 3
20 days. In some embodiments, in step (b), the cells are cultured for between 2 and 20 days. In
some embodiments, in step (b), the cells are cultured for between 2 and 10 days. In some
embodiments, in step (b), the cells are cultured for between 2 and 6 days. In some
embodiments, in step (b), the cells are cultured for between 2 and 4 days. In some
embodiments, in step (b), the cells are cultured for about 3 days.
25
In some embodiments, before step (a), the cells are cultured as a monolayer at an initial
density of at least 20000 cells/cm2. In some embodiments, before step (a), the cells are
cultured as a monolayer at an initial density of at least 100000 cells/cm2. In some
embodiments, before step (a), the cells are cultured as a monolayer at an initial density of
between 20000 and 1000000 cells/cm230 . In some embodiments, before step (a), the cells are
cultured as a monolayer at an initial density of between 100000 and 500000 cells/cm2. In
some embodiments, before step (a), the cells are cultured as a monolayer at an initial density
of about 240000 cells/cm2.
35 In some embodiments, in step (c), the cells are replated at a density of at least 1000
cells/cm2. In some embodiments, in step (c), the cells are replated at a density of at least
10000 cells/cm2. In some embodiments, in step (c), the cells are replated at a density of at
14
least 20000 cells/cm2. In some embodiments, in step (c), the cells are replated at a density of
at least 100000 cells/cm2. In some embodiments, in step (c), the cells are replated at a
density of between 20000 and 5000000 cells/cm2. In some embodiments, in step (c), the
cells are replated at a density of between 100000 and 1000000 cells/cm2. In some
embodiments, in step (c), the cells replated at a density of about 500000 cells/cm2. In 5 n some
embodiments, in step (c), the cells are replated on fibronectin, matrigel® or Cellstart®.
In some embodiments, the invention relates to a method for producing RPE cells comprising
steps (a), (b) and (c) disclosed above and further comprising the following steps:
10 (d) culturing the replated cells of step (c) in the presence of an activin pathway activator;
(e) replating the cells of step (d); and,
(f) culturing the replated cells of step (e).
In some embodiments, the activin pathway activator is activin A pathway activator. In some
15 embodiments, the activin pathway activator comprises activin A or activin B. In a preferred
embodiment, the activin pathway activator is activin A.
In some embodiments, in step (d), the cells are cultured in the presence of activin pathway
activator for at least 1 day. In some embodiments, in step (d), the cells are cultured in the
20 presence of activin pathway activator for at least 3 days. In some embodiments, in step (d),
the cells are cultured in the presence of activin pathway activator for between 1 and 50 days,
3 and 30 days or 3 and 20 days.
In some embodiments, in step (d), the cells are cultured in the presence of activin pathway
25 activator for at least 1 day and the cells are further cultured without the activin pathway
activator for at least 3 days. In some embodiments, in step (d), the cells are cultured in the
presence of activin pathway activator for at least 3 days and the cells are further cultured
without the activin pathway activator for at least 4 days. In some embodiments, in step (d),
the cells are cultured in the presence of activin pathway activator for between 1 and 10 days
30 and the cell are further cultured without the activin pathway activator for between 5 and 30
days. In some embodiments, in step (d), the cells are cultured in the presence of activin
pathway activator for about 3 days and the cell are further cultured without the activin
pathway activator for between 5 and 30 days.
35 In some embodiments, in step (d), the concentration of activin pathway activator is between
1ng/mL and 10μg/mL. In some embodiments, in step (d), the concentration of activin
pathway activator is between 1ng/mL and 1μg/mL. In some embodiments, in step (d), the
15
concentration of activin pathway activator is between 10ng/mL and 500ng/mL. In some
embodiments, in step (d), the activin pathway activator is activin A at a concentration of
about 100ng/mL.
In some embodiments, in step (e), the cells are replated at a density of at least 1005 0
cells/cm2. In some embodiments, in step (e), the cells are replated at a density of at least
20000 cells/cm2. In some embodiments, in step (e), the cells are replated at a density of at
least 100000 cells/cm2. In some embodiments, in step (e), the cells are replated at a density
of between 20000 and 5000000 cells/cm2. In some embodiments, in step (e), the cells are
replated at a density of between 20000 and 1000000 cells/cm210 . In some embodiments, in
step (e), the cells are replated at a density of between 20000 and 500000 cells/cm2. In some
embodiments, in step (e), the cells are replated at a density of about 200000 cells/cm2. In
some embodiments, in step (e), the cells are replated on fibronectin, matrigel® or Cellstart®.
15 In some embodiments, in step (f), the cells are cultured for at least 5 days. In some
embodiments, in step (f), the cells are cultured for at least 7 days, at least 14 days or at least
21 days. In some embodiments, in step (f), the cells are cultured for at least 14 days. In
some embodiments, in step (f), the cells are cultured for between 5 and 40 days. In some
embodiments, in step (f), the cells are cultured for between 10 and 35 days. In some
20 embodiments, in step (f), the cells are cultured for between 21 and 35 days. In some
embodiments, in step (f), the cells are cultured for about 28 days.
In some embodiments, in step (d), the cells are cultured in the presence of cAMP, preferably
at a concentration between 0.01mM to 1M. In some embodiments, in step (d), the cells are
25 cultured in the presence of 0.1mM to 5mM cAMP. In some embodiments, in step (d), the
cells are cultured in the presence of 0.5mM cAMP.
In some embodiments, in step (f), the cells are cultured in the presence of cAMP, preferably
at a concentration between 0.01mM to 1M. In some embodiments, in step (f), the cells are
30 cultured in the presence of 0.1mM to 5mM cAMP. In some embodiments, in step (f), the cells
are cultured in the presence of 0.5mM cAMP.
The present disclosure also includes methods where the above disclosed embodiments of
steps (a), (b), (c), (d), (e) and/or (f) are combined.
35
In a preferred embodiment, the invention relates to a method for producing retinal pigment
epithelial cells comprising the following steps:
16
(a) culturing human ESCs or human iPSCs in the presence of 500nM to 2μM LDN193189
and 5μM to 20μM SB-431542 for between 3 and 5 days;
(b) culturing the cells of step (a) in the presence of 50 ng/mL to 500ng/mL of BMP2/6
heterodimer, BMP4/7 heterodimer or BMP3/8 heterodimer and in the absence of LDN193189
and SB-431542 for between 2 and 6 days; an5 d,
(c) replating the cells of step (b) at a density of between 100000 and 1000000 cells/cm2.
(d) culturing the replated cells of step (c) in the presence of about 10ng/mL to 500ng/mL
activin A for between 3 and 30 days;
(e) replating the cells of step (d) at a density of between 20000 and 500000 cells/cm2; and,
10 (f) culturing the replated cells of step (e) for between 10 and 35 days.
Late replating
In an alternative embodiment (late replating embodiment), the method for producing RPE
15 cells comprises the following steps:
(a) culturing pluripotent cells in the presence of a first SMAD inhibitor and a second SMAD
inhibitor;
(b) culturing the cells of step (a) in the presence of a BMP pathway activator and in the
absence of the first and second SMAD inhibitors; and then,
20 culturing said cells for at least 10 days in the absence of the BMP pathway activator;
(c) replating the cells of step (b) having a cobblestone morphology; and,
(d) culturing the replated cells of step (c).
The embodiments disclosed above in connection with steps (a), (b) and (c) of the early
25 replating embodiment are also embodiments of steps (a), (b) and (c) of the late replating
embodiment.
In some embodiments, in step (b), the cells are cultured for at least 20 days in the absence
of the BMP pathway activator. In some embodiments, in step (b), the cells are cultured for at
30 least 30 days in the absence of the BMP pathway activator. In some embodiments, in step
(b), the cells are cultured for at least 40 days in the absence of the BMP pathway activator. In
some embodiments, in step (b), the cells are cultured for between 10 and 60 days in the
absence of the BMP pathway activator. In some embodiments, in step (b), the cells are
cultured for between 30 and 50 days in the absence of the BMP pathway activator. In some
35 embodiments, in step (b), the cells are cultured for about 40 days in the absence of the BMP
pathway activator.
17
In some embodiments, in step (c), the cells are replated at a density of at least 1000
cells/cm2. In some embodiments, in step (c), the cells are replated at a density of at least
20000 cells/cm2. In some embodiments, in step (c), the cells are replated at a density of at
least 100000 cells/cm2. In some embodiments, in step (c), the cells are replated at a density
of between 20000 and 5000000 cells/cm2. In some embodiments, in step (c), the cells 5 s are
replated at a density of between 50000 and 1000000 cells/cm2. In some embodiments, in
step (c), the cells are replated at a density of between 50000 and 500000 cells/cm2. In some
embodiments, in step (c), the cells are replated at a density of about 200000 cells/cm2.
10 In some embodiments, in step (d), the cells are cultured for at least 3 days. In some
embodiments, in step (d), the cells are cultured for at least 5 days. In some embodiments, in
step (d), the cells are cultured for at least 10 days. In some embodiments, in step (d), the
cells are cultured for at least 14 days. In some embodiments, in step (d), the cells are
cultured for between 10 and 40 days. In some embodiments, in step (d), the cells are
15 cultured for between 10 and 20 days. In some embodiments, in step (d), the cells are
cultured for about 14 days.
In some embodiments, in step (d), the cells are cultured in the presence of cAMP, preferably
at a concentration between 0.01mM to 1M. In some embodiments, in step (d), the cells are
20 cultured in the presence of 0.1mM to 5mM cAMP. In some embodiments, in step (d), the
cells are cultured in the presence of 0.5mM cAMP.
In some embodiments, the method further comprises the following additional steps:
(e) replating the cells of step (d);
25 (f) culturing the replated cells of step (e).
In some embodiments, in step (e), the cells are replated at a density of at least 1000
cells/cm2. In some embodiments, in step (e), the cells are replated at a density of at least
20000 cells/cm2. In some embodiments, in step (e), the cells are replated at a density of at
least 100000 cells/cm230 . In some embodiments, in step (e), the cells are replated at a density
of between 20000 and 5000000 cells/cm2. In some embodiments, in step (e), the cells are
replated at a density of between 50000 and 1000000 cells/cm2. In some embodiments, in
step (e), the cells are replated at a density of between 50000 and 500000 cells/cm2. In some
embodiments, in step (e), the cells replated at a density of about 200000 cells/cm2.
35
In some embodiments, in step (f), the cells are cultured for at least 10 days. In some
embodiments, in step (f), the cells are cultured for at least 14 days. In some embodiments, in
18
step (f), the cells are cultured for at least 20 days. In some embodiments, in step (f), the cells
are cultured for at least 25 days. In some embodiments, in step (f), the cells are cultured for
at least 40 days. In some embodiments, in step (f), the cells are cultured for between 10 and
60 days. In some embodiments, in step (f), the cells are cultured for between 15 and 40
days. In some embodiments, in step (f), the cells are cultured for about 28 day5 s.
The present disclosure also includes methods where the above disclosed embodiments of
steps (a), (b), (c), (d), (e) and/or (f) are combined.
10 In a preferred embodiment, the invention relates to a method for producing RPE cells
comprising the following steps:
(a) culturing human ESCs or human iPSCs in the presence of 500nM to 2μM LDN193189
and 5μM to 20μM SB-431542 for between 3 and 5 days;
(b) culturing the cells of step (a) in the presence of 50 ng/mL to 500ng/mL of BMP2/6
15 heterodimer, BMP4/7 heterodimer or BMP3/8 heterodimer and in the absence of LDN193189
and SB-431542 for between 2 and 6 days; and then,
culturing said cells for between 30 and 50 days in the absence of the BMP pathway activator
(c) replating the cells of step (b) having a cobblestone morphology at a density of between
50000 and 500000 cells/cm2; and,
20 (d) culturing the replated cells of step (c) for between 10 and 20 days;
(e) replating the cells of step (d) at a density of between 50000 and 500000 cells/cm2; and,
(f) culturing the replated cells of step (e) for between 15 and 40 days.
The RPE cells prepared by the methods disclosed herein (including early replating and late
25 replating) can be harvested by various methods known to the skilled person. For example,
the RPE cells can be harvested by mechanical dissection or by dissociation with an enzyme
such as papain or trypsin.
The RPE cells prepared by the methods disclosed herein can be further purified, for example
30 without limitation, by techniques such as Fluorescence Activated Cell Sorting (FACS) or
Magnetic Activated Cell Sorting (MACS). These techniques involve the use of antibodies
against RPE-specific cell surface proteins (positive selection). In a preferred embodiment,
said RPE specific cell surface protein is CD59. For FACS, RPE cells can be labelled with
fluorophore conjugated antibodies targeting specific RPE cell surface markers. These
35 labelled cells can be purified using a cytometer to give rise to a highly homogeneous and
purified RPE population free of any contaminating cell type. Similarly in MACS, RPE cells
can be labelled with antibodies conjugated to magnetic nanoparticles and further purified by
19
application of magnetic field. Negative selection can also be applied by using antibodies
targeting potential contaminating cell types which would lead to their removal and also
contribute to generation of pure RPE population.
In some embodiments, the method for producing RPE cells disclosed herein comprises 5 ses a
purification step for enriching the cell population in cells expressing CD59. Enriching the cell
population in cells expressing CD59 is a means to enrich for mature RPE cells and remove
residual contaminating cells such as pluripotent cells and/or RPE progenitors that may
possibly be present in the final RPE cell population.
10 In some embodiments, the method for producing RPE cells disclosed herein comprises a
purification step comprising:
- contacting the cells with an anti-CD59 antibody conjugated to a fluorophore, and,
- selecting the cells that bind to the anti-CD59 antibody using FACS.
In a preferred embodiment, the anti-CD59 antibody is antibody Cat# 560747 (BD
15 Biosciences).
In some embodiments, the method for producing RPE cells disclosed herein comprises a
purification step as disclosed in Example 13b.
In some embodiments, the method for producing RPE cells disclosed herein comprises a
purification step comprising:
20 - contacting the cells with an anti-CD59 antibody conjugated to a magnetic particle, and,
- selecting the cells that bind to the anti-CD59 antibody using MACS.
Commercially available anti-CD59 antibody such as for example antibody Cat# 560747 (BD
Biosciences) can be used in the present invention.
25
In some embodiments, a purification step as disclosed above is performed after step (e) of
the early replating method. In some embodiments, a purification step as disclosed above is
performed after step (f) of the early replating method. In some embodiments, a purification
step as disclosed above is performed after step (c) of the late replating method. In some
30 embodiments, a purification step as disclosed above is performed after step (d) of the late
replating method.
In some embodiments, the invention relates to a method for producing RPE cells comprising:
35 a) providing a population of pluripotent cells;
b) inducing the differentiation of pluripotent cells into RPE cells, and,
c) enriching the cell population for cells expressing CD59.
20
In some embodiments, the invention relates to a method for producing RPE cells comprising:
a) providing a population of pluripotent cells;
b) inducing the differentiation of pluripotent cells into RPE cells, and,
c) enriching the cell population for cells expressing CD59 by
- contacting the cells with an anti-CD59 antibody conjugated to a fluorophore, an5 d,
- selecting the cells that bind to the anti-CD59 antibody using FACS.
In some embodiments, the invention relates to a method for producing RPE cells comprising:
a) providing a population of pluripotent cells;
b) inducing the differentiation of pluripotent cells into RPE cells, and,
10 c) enriching the cell population for cells expressing CD59 by
- contacting the cells with an anti-CD59 antibody conjugated to a magnetic particle, and,
- selecting the cells that bind to the anti-CD59 antibody using MACS.
In step b, the differentiation of pluripotent cells in RPE cells can be performed according to
any method known to the skilled person such as for example spontaneous differentiation or
15 directed differentiation methods. In particular, in step b, the differentiation of pluripotent cells
into RPE cells can be performed according to any method disclosed in WO08/129554,
WO09/051671, WO2011/063005, US2011269173, US20130196369, WO2013/184809,
WO08/087917, WO2011/028524 or WO2014/121077, which are incorporated herein by
reference.
20
In some embodiments, the invention relates to a method for purifying RPE cells comprising:
a) providing a cell population comprising RPE cells and non RPE cells;
b) increasing the percentage of RPE cells in the cell population by enriching the cell
population for cells expressing CD59.
25 In some embodiments, the invention relates to a method for purifying RPE cells comprising:
a) providing a cell population comprising RPE cells and non RPE cells;
b) increasing the percentage of RPE cells in the cell population by
- contacting the cell population with an anti-CD59 antibody conjugated to a
fluorophore, and,
30 - selecting the cells that bind to the anti-CD59 antibody using FACS.
In some embodiment, the invention relates to a method for purifying RPE cells comprising:
a) providing a cell population comprising RPE cells and non RPE cells;
b) increasing the percentage of RPE cells in the cell population by
- contacting the cell population with an anti-CD59 antibody conjugated to a magnetic
35 particle, and,
- selecting the cells that bind to the anti-CD59 antibody using MACS.
In some embodiments, non RPE cells are pluripotent cells or RPE progenitors.
21
In some embodiments, the term “RPE progenitors” refers to cells derived from pluripotent
cells such as hESC induced to differentiate into RPE cells but which have not fully completed
the differentiation process. In some embodiments, such “RPE progenitor” comprises one or
more morphological and functional attributes of an adult RPE cell and lacks at least on5 e
morphological and functional attributes of an adult RPE cells. In some embodiment, the RPE
progenitor expresses one or more of OCT4, NANOG or LIN28.
In some embodiments of the methods disclosed herein, the cells are cultured in a two10
dimensional culture under adhesion conditions, such as, for example, plate culture. In a
preferred embodiment, the cells are cultured as a monolayer. In some embodiments, the
cells are cultured on a cell-supporting substance, such as, for example without limitation,
collagen, gelatin, poly-L-Iysine, poly-D-Iysine, laminin, fibronectin, vitronectin, Cellstart®,
BME pathclear®, or Matrigel® (Becton, Dickinson and Company). In some embodiments,
15 the cells are cultured as a monolayer, for example, on collagen, gelatin, poly-L-Iysine, poly-
D-Iysine, laminin, fibronectin, vitronectin, Cellstart®, BME pathclear®, or Matrigel®. In a
preferred embodiment, the cells are cultured as a monolayer on Matrigel®. In a preferred
embodiment, the cells are cultured as a monolayer on fibronectin or vitronectin.
20 In some embodiments, some steps of the methods disclosed herein may be performed in a
three-dimensional culture under non-adhesion conditions, such as suspension culture. In
suspension culture, a majority of cells freely float as single cells, cell clusters and or as cell
aggregates in a liquid medium. The cells can be cultured in a three dimensional system
according to method known to the skilled person (see for example Keller et aI, Current
25 Opinion in Cell Biology, Vol 7 (6), 862-869 (1995)) or Watanabe et aI., Nature Neuroscience
8, 288-296 (2005)).
In some embodiments, some steps of the methods disclosed herein are carried out in a three
dimensional culture such as, for example without limitation, suspension culture and some
30 steps are carried out in a two dimensional culture (e.g. cells cultured as a monolayer). In
some embodiments, step (a) and/or (b) are carried out in a suspension culture and the
following steps are carried out in a two dimensional culture (e.g. cells cultured as a
monolayer).
35 In some embodiments, the cells are incubated with a Rho-associated protein kinase (ROCK)
inhibitor before being plated. In some embodiments, the cells are incubated with a ROCK
inhibitor before step (a). The ROCK inhibitor is a substance permitting survival of dissociated
22
human embryonic stem cells (see K. Watanabe et Al., Nat. Biotech., 25: 681-686 (2007)).
Examples of ROCK inhibitors which can be used in the method of the invention are, without
limitation, Y-27632, H-1152, Y-30141, Wf-536, HA-1077, GSK269962A and SB-772077-B. In
some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, before step (a),
the pluripotent cells are plated in the presence of a ROCK inhibitor. In some embodiments5 ,
the cells are cultured in the presence of a ROCK inhibitor for 1 or 2 days post plating. In
some embodiments, the first replating of the method of the invention is carried out in the
presence of a ROCK inhibitor. In some embodiments, the cells are cultured in the presence
of a ROCK inhibitor for 1 or 2 days post first replating.
10
In the methods of the invention, the cell can be cultured in any basic medium suitable for the
culture of pluripotent cells, preferably human pluripotent cells. In some embodiments, the
cells are cultured in a basic medium suitable for the culture of human embryonic stem cells.
15
Examples of suitable basic media include, without limitation, IMDM medium, medium 199,
Eagle's Minimum Essential Medium (EMEM), AMEM medium, Dulbecco's modified Eagle's
Medium (DMEM), KO-DMEM, Ham's F12 medium, RPMI 1640 medium, Fischer's medium,
Glasgow MEM, TesR1, TesR2, Essential 8 and mixtures thereof. In some embodiments the
20 medium comprises serum. In some embodiments, the medium is serum free. In a preferred
embodiment, the basic medium is TesR1 or TesR2.
The medium may further contain, if desirable, one or more serum substitutes, such as for
example albumin, transferrin, Knockout Serum Replacement (KSR), fatty acid, insulin, a
25 collagen precursor, trace elements, 2-mercaptoethanol, 3' –thiol, glycerol, B27-supplement,
and N2-supplement, as well as one or more substances such as, lipids, amino acids,
nonessential amino acids, vitamins, growth factors, cytokines, antibiotics, antioxidants,
pyruvate, a buffering agent, and inorganic salts.
30 The basic medium used for the cell culture in the method of the invention can be
supplemented as appropriate with, for example without limitation, SMAD inhibitors, BMP
pathway activators, activin pathway activators and/or cAMP.
In some embodiments of the above disclosed methods, the cells used in step (a) are hESC
35 or human IPSc and the method is carried out under xeno-free conditions, i.e without using
any animal derived material other than human. For example, when the method is carried out
23
under xeno-free conditions, the medium and the cell supporting substance do not comprise
any animal derived material other than human.
In some embodiments, replating comprises dissociating the plated cells, preferably
dissociating the monolayer of cells, and plating the dissociated cells. Preferably, the cells 5 s are
dissociated using an enzyme such as for example trypsin, collagenase IV, collagenase I,
dispase or a commercially available cell dissociation buffer. Preferably, the cells are
dissociated using TrypLE Select®.
10 In some embodiments, the RPE cells obtained or obtainable by the methods disclosed herein
are further expanded. In some embodiments the expansion step is carried out in a two
dimensional culture, under adhesion conditions. In some embodiments, the expansion step
comprises:
- replating RPE cells; and,
15 - culturing the replated RPE cells.
In some embodiments, the RPE cells are replated on a cell supporting substance. Suitable
cell supporting substances include, for example without limitation, collagen, gelatin, poly-LIysine,
poly-D-Iysine, laminin, fibronectin, vitronectin, Cellstart®, Matrigel® or BME
pathclear® (BME PathClear® is a soluble form of basement membrane purified from
20 Engelbreth-Holm-Swarm (EHS) tumor. It is mainly comprised of laminin, collagen IV,
entactin, and heparin sulfate proteoglycan). In a preferred embodiment, the cell supporting
substance is selected from Matrigel®, Fibronectin or Cellstart®, preferably Cellstart®.
In some embodiments, the RPE cells are replated at a density between 1000 and 100000
cells/cm225 . In some embodiments, the RPE cells are replated at a density between 5000 and
100000 cells/cm2. In some embodiments, the RPE cells are replated at a density between
10000 and 40000 cells/cm2. In some embodiments, the RPE cells are replated at a density
between 10000 and 30000 cells/cm2. In some embodiments, the RPE cells are replated at a
density of about 20000 cells/cm2.
30
In some embodiments, the replated cells are cultured for at least 7 days. In some
embodiments, the replated cells are cultured for at least 14 days. In some embodiments, the
replated cells are cultured for at least 28 days. In some embodiments, the replated cells are
cultured for at least 42 days. In some embodiments, the replated cells are cultured for
35 between 21 days and 70 days. In some embodiments, the replated cells are cultured for
between 30 days and 60 days. In some embodiments, the replated cells are cultured for
about 49 days.
24
In some embodiments, RPE cells are cultured in the presence of cAMP, preferably at a
concentration between 0.01mM to 1M. In some embodiments, RPE cells are cultured in the
presence of 0.1mM to 5mM cAMP. In some embodiments, RPE cells are cultured in the
presence of about 0.5mM cAMP5 .
In some embodiments, RPE cells are cultured in the presence of an agent which increases
the intracellular concentration of cAMP. In some embodiments, said agent is an Adenyl
Cyclase activator, preferably forskolin. In some embodiments, said agent is a
10 phosphodiesterase (PDE) inhibitor, preferably a PDE1, PDE2, PDE3, PDE4, PDE7, PDE8,
PDE10 and/or PDE11 inhibitor. In some embodiments, said agent is a PDE4, PDE7 and/or
PDE8 inhibitor.
In some embodiments, RPE cells are cultured in the presence of a SMAD inhibitor,
15 preferably at a concentration between 1nM to 100μM. In some embodiments, RPE cells are
cultured in the presence of 10nM to 10μM SMAD inhibitor. In some embodiments, RPE cells
are cultured in the presence of about 10nM to 1μM SMAD inhibitor. In some embodiments,
said SMAD inhibitor is an inhibitor of TGF type I receptor (ALK5) and/or TGF type II
receptor. In a preferred embodiment, said SMAD inhibitor is an ALK5 inhibitor. In some
20 embodiments, said inhibitor is 2-(6-methylpyridin-2-yl)-N-(pyridin-4-yl)quinazolin-4-amine, 6-
(1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)quinazolin-4(3H)-one, or 4-methoxy-6-(3-(6-
methylpyridin-2-yl)-1H-pyrazol-4-yl)quinoline. Examples of SMAD inhibitors that can be used
in the present invention can also be found for example in EP2409708A1 or in Yingling JM et
al. Nature Reviews/Drug Discovery Vol. 3:1011-1022 (2004).
25
In some embodiments, RPE cells are cultured in the presence of cAMP or an agent which
increases the intracellular concentration of cAMP, preferably cAMP, and the yield of the
expansion step is increased as compared to similar conditions without said agent or cAMP.
30 The invention also relates to a method for expanding RPE cells comprising the step of
culturing said RPE cells in the presence of SMAD inhibitor, cAMP or an agent which
increases the intracellular concentration of cAMP. In some embodiments, the invention
relates to a method for expanding RPE cells comprising the following steps:
(a) plating RPE cells at a density of at least 1000 cells/cm2, and,
35 (b) culturing said RPE cells in the presence of SMAD inhibitor, cAMP or an agent which
increases the intracellular concentration of cAMP.
25
In some embodiments, in step (a), the RPE cells are plated on a cell supporting substance
for example selected from collagen, gelatin, poly-L-Iysine, poly-D-Iysine, laminin, fibronectin,
vitronectin Cellstart®, Matrige® or BME pathclear®. In a preferred embodiment, in step (a),
the cell supporting substance is selected from Matrigel®, Fibronectin or Cellstart®, preferably
Cellstart5 ®.
In some embodiments, in step (a), the RPE cells are plated at a density between 1000 and
100000 cells/cm2. In some embodiments, in step (a), the RPE cells are plated at a density
between 5000 and 100000 cells/cm2. In some embodiments, in step (a), the RPE cells are
plated at a density between 10000 and 40000 cells/cm210 . In some embodiments, in step (a),
the RPE cells are plated at a density between 10000 and 30000 cells/cm2. In some
embodiments, in step (a), the RPE cells are plated at a density of about 20000 cells/cm2.
In some embodiments, in step (b), the RPE cells are cultured for at least 7 days. In some
15 embodiments, the replated cells are cultured for at least 14 days. In some embodiments, in
step (b), the replated cells are cultured for at least 28 days. In some embodiments, in step
(b), the replated cells are cultured for at least 42 days. In some embodiments, in step (b), the
replated cells are cultured for between 21 days and 70 days. In some embodiments, in step
(b) the replated cells are cultured for between 30 days and 60 days. In some embodiments
20 the replated cells are cultured for about 49 days.
In some embodiments, in step (b), RPE cells are cultured in the presence of an agent which
increases the intracellular concentration of cAMP. In some embodiments, said agent is an
Adenyl Cyclase activator, preferably forskolin. In some embodiments, said agent is a
25 phosphodiesterase (PDE) inhibitor, preferably a PDE1, PDE2, PDE3, PDE4, PDE7, PDE8,
PDE10 and/or PDE11 inhibitor. In some embodiments, said agent is a PDE4, PDE7 and/or
PDE8 inhibitor.
In some embodiments, in step (b), RPE cells are cultured in the presence of cAMP,
30 preferably at a concentration between 0.01mM to 1M. In some embodiments, in step (b),
RPE cells are cultured in the presence of 0.1mM to 5mM cAMP. In some embodiments, in
step (b), RPE cells are cultured in the presence of about 0.5mM cAMP.
In some embodiments, in step (b), RPE cells are cultured in the presence of cAMP or an
35 agent which increases the intracellular concentration of cAMP, preferably cAMP, and the
yield of the method for expanding RPE cells is increased as compared to the same method
without said agent or cAMP.
26
In some embodiments, in step (b), RPE cells are cultured in the presence of a SMAD
inhibitor, preferably at a concentration between 1nM to 100μM. In some embodiments, RPE
cells are cultured in the presence of 10nM to 10μM SMAD inhibitor. In some embodiments,
RPE cells are cultured in the presence of about 10nM to 1μM SMAD inhibitor. In som5 e
embodiments, said SMAD inhibitor is an inhibitor of TGF type I receptor (ALK5) and/or
TGF type II receptor. In a preferred embodiment, said SMAD inhibitor is an ALK5 inhibitor.
In some embodiments, said inhibitor is 2-(6-methylpyridin-2-yl)-N-(pyridin-4-yl)quinazolin-4-
amine, 6-(1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)quinazolin-4(3H)-one, or 4-methoxy-6-(3-
10 (6-methylpyridin-2-yl)-1H-pyrazol-4-yl)quinoline. Examples of SMAD inhibitor that can be
used in the present invention can also be found for example in EP2409708A1 or in Yingling
JM et al. Nature Reviews/Drug Discovery Vol. 3:1011-1022 (2004).
15 In some embodiments, the invention relates to RPE cells obtained by a method disclosed
herein. In some embodiments, the invention relates to RPE cells obtainable by a method
disclosed herein.
The RPE cells obtained or obtainable by the methods disclosed herein can be used as a
20 research tool. For example, the RPE cells can be used in in vitro models for the development
of new drugs to promote their survival, regeneration and/or function or for high throughput
screening for compounds that have a toxic or regenerative effect on RPE cells.
The RPE cells obtained or obtainable by the methods disclosed herein can be used in
25 therapy. In some embodiments, the RPE cells can be used for the treatment of retinal
diseases.
In some embodiments, the RPE cells are formulated in a pharmaceutical composition
suitable for transplantation into the eye of a subject affected with a retinal disease.
30
In some embodiments, the pharmaceutical composition suitable for transplantation into the
eye comprises a structure suitable for supporting RPE cells and RPE cells. Non limitative
examples of such pharmaceutical compositions are disclosed in WO2009/127809,
WO2004/033635 or WO2012/009377 or WO2012177968, which are herein incorporated by
35 reference in their entirety.
27
In a preferred embodiment the pharmaceutical composition comprises a porous membrane
and RPE cells. In some embodiments, the pores of the membrane are between 0.2μm and
0.5μm in diameter and the pore density is between 1x107 and 3x108 pores per cm2. In some
embodiments the membrane is coated on one side with a coating supporting RPE cells. In
some embodiments, the coating comprises a glycoprotein, preferably selected from lamini5 n
or vitronectin. In a preferred embodiment, the coating comprises vitronectin. In some
embodiments, the membrane is made of polyester.
In an alternative embodiment, the pharmaceutical composition comprises RPE cells in
10 suspension in a medium suitable for transplantation into the eye of the subject. Examples of
such pharmaceutical compositions are disclosed in WO2013/074681, which is herein
incorporated by reference in its entirety.
The RPE cells obtained by the method disclosed herein may be transplanted to various
15 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 (between the photoreceptor outer
segments and the choroids). In addition, transplantation into additional ocular compartments
can be considered including the vitreal space, the inner or outer retina, the retinal periphery
and within the choroids.
20
Transplantation of RPE cells into the eye can be performed by various techniques known in
the art (see for example US patents No 5962027, 6045791 and 5,941,250, which are herein
incorporated by reference in their entirety).
25 In some embodiments, transplantation is performed via pars plana vitrectomy surgery
followed by delivery of the cells through a small retinal opening into the sub-retinal space. In
some embodiments, the RPE cells are transplanted into the eye using a suitable device (see
for example WO2012/099873 or WO2012/004592, which are herein incorporated by
reference in their entirety).
30
In some embodiments, the transplantation is performed by direct injection into the eye of the
subject.
In some embodiments, the RPE cells obtained by the methods disclosed herein can be used
35 for the treatment of retinal diseases. In some embodiments, the invention relates to RPE
cells obtained or obtainable by the methods disclosed herein or a pharmaceutical
composition comprising such cells for use in the treatment of retinal disease in a subject. In
28
some embodiments, the invention relates to the use of RPE cells obtained or obtainable by
the methods disclosed herein or a pharmaceutical composition comprising such cells for the
manufacture of a medicament for the treatment of retinal disease in a subject. In some
embodiments, the invention relates to a method for the treatment of a retinal disease in a
subject by administering RPE cells obtained or obtainable by the methods disclosed herei5 n
or a pharmaceutical composition comprising such cells to said subject.
In some embodiments, the subject is a mammal, preferably a human.
10 In some embodiments, the retinal disease is a disease associated with retinal dysfunction,
retinal injury, and/or loss or degradation of retinal pigment epithelium. In some embodiments,
the retinal disease is selected from retinitis pigmentosa, leber’s congenital amaurosis,
hereditary or acquired macular degeneration, age related macular degeneration (AMD), Best
disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy as well as
15 other dystrophies of the RPE cells, diabetic retinopathy or Stargardt disease. In a preferred
embodiment, retinal disease is retinitis pigmentosa or age related macular degeneration
(AMD). In a preferred embodiment, the retinal disease is age related macular degeneration.
Examples
20
Example 1 – Directed differentiation with early replating
All work was carried out in a sterile tissue culture hood. Shef-1 hESC were routinely cultured
on Matrigel (BD) in TeSR1 media (Stem Cell Technologies). WA26 hESC (Wicell) were
25 routinely cultured in Essential 8 Medium (Life Technologies) on human vitronectin (Life
Technologies). Cultures were passaged twice per week using 0.5mM EDTA solution (Sigma)
to dissociate the colonies into smaller aggregates, which were then replated in medium
containing 10μM Y-27632 (Rho-associated kinase inhibitor) (Sigma). The culture medium
was replaced daily.
30
Shef1 or WA26 hESC (Wicell) were incubated with 10μM Y276352 (ROCK inhibitor) for
35min at 37oC. Media was removed and the cells were washed with 5ml PBS (-MgCl2, -
CaCl2) (hereafter PBS (-/-)). 2mL TrypLE select® was added and cells incubated at 37oC/5%
CO2 in a humidified incubator for 6-8min. DMEM KSRXF media was prepared as follows:
35
29
Component Catalogue Number Volume (mL)
Knockout (KO) DMEM 10829-018 (Life Technologies) 308
Xeno-free Knockout Serum
Replacement
12618-012 (Life Technologies) 80
Glutamax I 35050 (Life Technologies) 4
2-mercaptoethanol (70uL
diluted in 100mL KO DMEM)
M3148 (Sigma) 4
Non-essential amino acids 11140-035 (Life Technologies) 4
TesR2 complete media (TesR2) was prepared as follows:
Component Catalogue Number Volume (mL)
TesR2 basal media 05860 (Stem cell technologies) 78
TesR2 5x supplement 05860 (Stem cell technologies) 20
TesR2 250x supplement 05860 (Stem cell technologies) 0.4
5mL DMEM KSRXF media was added and pipetted up and down to achieve a single cell
suspension. The suspension was transferred to a 15mL falcon tube and centrifuged at 300x5 g
for 4min. The supernatant was aspirated and the pellet resuspended in 5mL TesR2 complete
media®. The cell suspension was passed through a 40μm cell strainer into a 50mL falcon
tube and the cell strainer was then washed with 1mL TesR2 complete media®. Cells were
centrifuged at 1300rpm for 4min. The supernatant was aspirated and the pellet resuspended
10 in 3mL TesR2 complete media® supplemented with 5μM Y276352. T25 flasks were coated
with the required matrix e.g. Matrigel or Fibronectin. Matrigel was thawed overnight in the
fridge and diluted 1:15 with Knockout DMEM before use. Fibronectin was diluted 1:10 in PBS
(-/-). 2.5 ml diluted matrix was used for coating a T25 flask and incubated for 3 hours at 37oC.
Cells were counted and plated in the coated culture vessel at the appropriate density to
obtain a monolayer. For a T25 flask, cells were seeded at a density of 240000 cells/cm2 15 in a
total volume of 10ml in TeSR2 comprising 5μM Y276352. This timepoint is designated as
Day 0. 24hours after plating (Day 1), media was aspirated and replaced with 10mL/flask of
TesR2 complete media (no Rock inhibitor). 48hours after plating (Day 2), media was
aspirated and replaced with 10mL/flask DMEM KSRXF media containing 1μM LDN193189
20 and 10μM SB-431542. The media comprising the two inhibitors was replenished everyday.
On Day 6, media was aspirated and replaced with 10mL/flask DMEM KSRXF containing
100ng/mL BMP4/7 heterodimer. Fresh media with BMP4/7 was replenished every day.
30
On Day 9, cells were replated as follows (Early Replate 1). First, culture vessels e.g T12.5
flasks, 96-well CellBind plates or 384-well CellBind plates were coated with the required
matrix e.g. Matrigel, Fibronectin or Cellstart. Matrigel was thawed overnight in the fridge and
diluted 1:15 with DMEM before use. Fibronectin was diluted 1:10 in PBS (-/-). Cellstart was
diluted 1:50 in PBS (+MgCl2, +CaCl2) (hereafter PBS (+/+)). 1.5 ml diluted matrix was use5 d
for coating a T12.5 flask and incubated for 3 hours at 37oC. Next, 10μM Y276352 was added
to each T25 flask of cells (at Day 9 of the differentiation protocol) and incubated at 37oC for
35min. Media was aspirated and cells were washed twice with 5mL PBS(-/-). 2.5mL TrypLE
select® was added to each flask and the flask transferred to 37oC for 15-25 min, until cells
10 had lifted from the flask. 5mL DMEM KSRXF media was added to each flask and used to
wash the surface of the flask. The cell suspension was passed through a 40μm cell strainer.
Cells were centrifuged at 400xg for 5min at room temperature. Supernatant was aspirated
and the pellet resuspended in 10mL DMEM KSRXF media (+ 5μM Y276352). Supernatant
was aspirated and the pellet resuspended in 10mL DMEM KSRXF media (5μM Y276352).
Cells were counted and plated into coated culture vessels at a density of 500000 cells/ cm215 .
24h after after replating (i.e at D10 which can also be noted D9-1 of the differentiation
protocol), the media was changed to DMEM KSRXF + 100ng/mL activin A. Media was
replenished with fresh activin A three times a week.
20 After D9-19 (i.e day D28), cells were replated to yield a homogeneous population of RPE
cells (Early Replate 2). The media was aspirated and cells washed 2x with 5mL PBS(-/-).
2.5mL Accutase was added to each flask and incubated at 37oC for about 35min, until cells
had lifted from the flask. 5mL DMEM KSRXF media was added to each flask and used to
wash the surface of the flask, before transferring the contents into a 50mL falcon tube
25 through a 70μm strainer. Cells were centrifuged at 400xg for 5min at room temperature. The
supernatant was aspirated and the pellet resuspended in 10mL DMEM KSRXF media. Cells
were counted using a haemocytometer and plated in DMEM KSRXF media in coated culture
vessels (e.g Cellstart 1:50 diluted in PBS (+/+)) at various densities e.g 120000/cm2. Fresh
media was replenished twice a week.
30 Cells were maintained in culture for 14 days. The resulting RPE cells were characterized
inter alia by testing for expression of RPE markers (PMEL17, ZO1, BEST1, CRALBP) by
immunocytochemistry and qPCR. More than 90% of the cells expressed the RPE marker
PMEL17.
35 This protocol led to generation of RPE cells which express the RPE marker PMEL17 as well
as other mature RPE markers such as CRALBP and MERTK.
31
This protocol involves treating a monolayer of pluripotent cells with SMAD inhibitors,
preferably LDN193189 and SB-431542 followed by activation of the BMP pathway for
example using a recombinant BMP4/7 heterodimer protein. Following LDN193189/SB-
431542 and BMP4/7 treatment, cells are replated (Early Replate 1) and can be treated with
activin A. Following treatment with activin A, cells can be replated for a second time (5 Early
Replate 2) into basal media and maintained in culture to obtain pure RPE cells cultures. This
leads to generation of homogeneous RPE cells cultures.
Without being bound to any theory, it is believed that the inhibition of the TGF signaling
10 using the SMAD inhibitors leads to differentiation of hESC towards anterior neuroectoderm
(ANE). Subsequent treatment with BMP pathway activators such as BMP4/7 induces
differentiation of the ANE towards eye field. The subsequent replating and optional treatment
with activin A led to a differentiation towards the RPE fate.
15 The present disclosure therefore provides a method for the robust and reproducible
differentiation of hESCs to give rise to pure RPE cells. In addition, this protocol is easily
scalable to give high yield. The above method can be used for reproducibly and efficiently
differentiate hESCs into RPE cells in xeno-free conditions.
20 Example 2 – Treatment with SMAD inhibitors
This example illustrates the effect of SMAD inhibitors on hESCs.
2.1. Treatment with SMAD inhibitors leads to ANE formation
25
Shef-1 hESCs were seeded onto Matrigel coated 96-well plates at a density of 125000
cells/cm2. On Day 2 post seeding, cells were treated with 1μM LDN193189 and 10μM SB-
431542 and samples were fixed at Day 2, Day 6, Day 8 and Day 10. Immunocytochemistry
was carried out for PAX6 (marker of ANE) expression and OCT4 (marker of pluripotent
30 hESCs) downregulation. A uniform induction of PAX6 protein and a uniform decrease of
OCT4 over the time course of differentiation was seen in samples induced with LDN193189
and SB-431542 (Figure 1B). This was observed not only on the whole surface of one well of
a 96-well plate but similarly in all the wells within the plate indicating a robust induction with
low inter/intra plate variability. In contrast, samples not treated with LDN193189 and SB-
35 431542 and maintained in media alone expressed low levels of PAX6 and higher levels of
OCT4 at the end of the timecourse indicating that efficient induction of ANE did not occur in
the absence of LDN193189 and SB-431542 (Figure 1C).
32
2.2. Treatment with SMAD inhibitors for two days
Shef-1 hESCs were seeded onto Matrigel coated 96-well plates at a density of 125005 0
cells/cm2. On Day 2 post seeding, cells were treated with 1μM LDN193189 and 10μM SB-
431542 for different lengths of time as described in Table 1.
Table 1
Day2- 0 Day 2-1 Day 2-2 Day 2-3 Day 2-4 Day2-5
Control+ LDN/SB LDN/SB LDN/SB LDN/SB LDN/SB LDN/SB
Control- DMEM KSRXF
DMEM
KSRXF
DMEM
KSRXF
DMEM
KSRXF
DMEM
KSRXF
DMEM
KSRXF
LDN/SB 2 day LDN/SB LDN/SB
DMEM
KSRXF
DMEM
KSRXF
DMEM
KSRXF
DMEM
KSRXF
10
Cells were immunostained for PAX6 and OCT4. The level of PAX6 upregulation and OCT4
downregulation was similar for all conditions tested (Figure 1C). This shows that at least 2
days of LDN193189/SB-431542 results in ANE induction.
15 Example 3 – Induction of RPE markers
Example 3.1. Induction of MITF by activation of BMP pathway
This example illustrates the effect of a BMP pathway activator on RPE marker expression.
20 Shef-1 hESCs were seeded onto Matrigel coated 96-well plates at a density of 125000
cells/cm2. On Day 2 post seeding 1μM LDN193189 and 10μM SB-431542 were applied for 4
days. Cells for the uninduced control were left untreated. On Day 6, 100 ng/ml BMP4/7 or
100ng/ml activin A + 10mM Nicotinamide or nothing was added to the media for 3 days. On
Day 9, BMP4/7 or activin A and Nicotinamide were withdrawn and cells were treated with
25 DMEM KSRXF alone for 4 days. Samples were prepared for RNA extraction and qPCR
analysis. The results are summarized in Figure 2A.
BMP4/7 induced expression of RPE genes e.g MITF and PMEL17 as compared to
uninduced or LDN193189/SB-431542 only treated controls. Furthermore, activin A +
30 Nicotinamide could not substitute for BMP4/7 (Figure 2A). Immunocytochemistry was also
performed on samples that were treated with LDN193189/SB-431542 followed by BMP4/7
33
which confirmed expression of RPE markers e.g MITF and PMEL17 (Figure 2B). These
results demonstrate that a BMP pathway activator strongly induces MITF expression and
PMEL17 expression.
Example 5 e 3.2
Shef-1 hESCs were treated with 1μM LDN193189 and 10μM SB-431542 from Day 2 to Day6
followed by 100ng/ml BMP4/7 from Day6 to Day9 (induced cells). Uninduced cells are
maintained without exposure to both LDN/SB and BMP4/7. Immunocytochemistry was
10 performed for PAX6, LHX2, OTX2, SOX11 and SOX2 which are markers known to be
expressed when cells are committed to the eye field fate. OCT4, a marker of pluripotency, is
downregulated from Day 2 to Day9 in induced cells. PAX6, LHX2, OTX2, SOX11 and SOX2
are upregulated from Day2 to Day9 and this upregulation is not achieved in uninduced
samples. This shows that the directed differentiation protocol induces cells towards an eye
15 field state which is then committed towards an RPE fate.
Example 4 – Use of alternative BMP pathway activators
This example illustrates the effect of various BMP pathway activators on RPE marker
20 expression.
Shef-1 hESCs were seeded onto Matrigel coated 96-well plates at a density of 125000
cells/cm2. On Day 2 post seeding, 1μM LDN193189 and 10μM SB-431542 were applied for 4
days. On Day 6, 50-200ng/ml BMP4/7 heterodimer or 200ng/ml BMP4, 300ng/ml BMP7,
25 100ng/ml BMP2/6 were added for a period of 3 days. On Day 9, BMPs were withdrawn and
cells maintained in DMEM KSRXF alone for 4 days. On Day 13, MITF expression was tested
by Immunostaining and qPCR analysis. Treatment with either BMP4/7 heterodimer or other
BMPs induced expression of MITF to a similar level (Figure 3). This showed that BMP4/7
could be substituted with other BMPs.
30
These results demonstrate that different BMP pathway activators can be used to induce
MITF expression.
Example 5 – First replating step
35
Shef-1 hESCs were seeded onto a Matrigel coated T25 flask at a density of 240000
cells/cm2. On Day 2 post seeding, 1μM LDN193189 and 10μM SB-431542 were applied for 4
34
days. On Day 6, 100 ng/ml BMP4/7 was added to the media for 3 days. Cells were replated
at either Day 6, Day 9 or Day 12 of the differentiation protocol into DMEM KSRXF alone or
DMEM KSRXF supplemented with either 100ng/ml activin A, 0.5mM cAMP or 100ng/ml
BMP4/7 at various densities. Cells replated at Day 6 were maintained for 3 days post
replating in DMEM KSRXF supplemented with 100ng/ml BMP4/7 before switching to activi5 n
A , cAMP or BMP4/7. Cells replated at Day 12 were maintained from Day 9 to Day 12 in
DMEM KSRXF alone before replating. Replated cells did not survive in the presence of
BMP4/7 and this condition was discarded from subsequent analysis. Mature RPE cells
sample obtained by spontaneous differentiation as disclosed in Example 10 (a) was used as
10 a control to compare the similarity between the populations obtained upon the first replating
step of directed differentiation and mature RPE cells. 19 days post replating, cells were fixed
for immunocytochemistry and samples were collected for qPCR. Immunocytochemistry with
mature RPE markers e.g CRALBP and MERTK showed that replating at D9 in the presence
of activin A was optimum and yielded high levels of RPE marker expression (Figure 4A and
15 4B). QPCR analysis with a panel of markers also indicated Day 9 to be the optimum time for
replating (Figure 4C, 4D and 4E). Similar results were obtained when cells were cultured on
different matrices e.g Matrigel, Cellstart or Fibronectin before and after replate.
Example 6 – Duration of exposure to activin A
20
This example illustrates the effects of activin A exposure duration on RPE differentiation.
WA26 hESCs (Wicell) were seeded onto Matrigel coated T25 flask at a density of 240000
cells/cm2. On Day 2 post seeding, 1μM LDN193189 and 10μM SB-431542 were applied for 4
25 days. On Day 6, 100ng/ml BMP4/7 was added to the media for 3 days. On Day 9, cells were
replated into 96 well CellBind plates coated with Matrigel or Cellstart at a density of 500000
cells/cm2. The cells were maintained in either DMEM KSRXF alone or DMEM KSRXF
supplemented with 100ng/ml activin A for different lengths of time e.g 3 days, 5 days, 10
days or 18 days. At D9-18, cells were fixed for immunostaining and stained for CRALBP, a
30 marker of RPE cells. The level of CRALBP expression was similar for all activin A treatments
tested (Figure 5). These results demonstrate that a short exposure to activin A is sufficient
for inducing RPE cells differentiation.
Example 7 – Second replating step at various densities
35
WA26 hESCs (Wicell) were seeded onto Matrigel coated T25 flask at a density of 240000
cells/cm2. On Day 2 post seeding, 1μM LDN193189 and 10μM SB-431542 were applied for 4
35
days. On Day 6, 100ng/ml BMP4/7 was added to the media for 3 days. On Day 9, cells were
replated into T12.5 flasks coated with either Matrigel or Cellstart at a density of 500000
cells/cm2. The cells were maintained in DMEM KSRXF supplemented with 100ng/ml activin A
for 19 days. At D9-19, cells were replated into Cellstart coated 96-well or 384-well plates at
various densities (Early Replate 2). The cells were maintained for 20 days in media alone 5 e or
media supplemented with 0.5mM cAMP. At D9-19-20, cells were fixed for immunostaining for
RPE markers. Both 96 and 384 well formats yielded similar results of >95% expression of
PMEL17 and about 60% expression of CRALBP (Figures 6 and 7). Furthermore, expression
of ZO1, another marker of mature RPE cells was confirmed by immunostaining.
10
Example 8 – Directed differentiation with late replating
The protocol up to Day 9 was identical to the protocol disclosed above in Example 1.
15 On Day 9, media was replaced with 10ml DMEM KSRXF per flask. The cells were
maintained in this media until Day 50 with fresh media change thrice a week. Around Day 50,
cobble-stoned cells were visible in the flask interspersed with other cells of different
morphologies. Also, the central area of the flask had a distinct morphology with several areas
of high density that had neuronal projections.
20 To carry out replating, media was removed from the flask and cells washed once with 5mL
PBS (-/-). 5ml PBS was added to the flask and the central dense area was scraped using a
cell scraper and discarded. The flask was washed again with 5 ml PBS (-/-). 5mL Accutase
was added to the flask and incubated at 37oC for about 50 min, until cells had lifted from the
flask. 5mL DMEM KSRXF media was added to each flask and used to wash the surface of
25 the flask, before transferring the contents into a 50mL falcon tube through a 70μm strainer.
Cells were centrifuged at 400xg for 5min at room temperature. The supernatant was
aspirated and the pellet resuspended in 10mL DMEM KSRXF media. Cells were counted
using a haemocytometer and plated in DMEM KSRXF media in coated culture vessels (e.g
Cellstart 1:50 diluted in PBS (+/+) at various densities e.g 200000/cm2. Fresh media was
30 replenished twice a week.
Cells were maintained in culture for 14 days. The resulting RPE cells were characterized by
testing for expression of RPE markers (PMEL17, ZO1, BEST1, CRALBP) by
immunocytochemistry and qPCR. The functionality of RPE cells was tested by analysing
35 secretion of VEGF and PEDF proteins which is an indicator of RPE cells maturity.
36
The present disclosure therefore provides a method for the robust and reproducible
differentiation of hESCs to give rise to RPE cells. In addition this protocol is easily scalable to
give high yield. The above method can be used for reproducibly and efficiently differentiate
hESCs into RPE cells in xeno-free free conditions
5
Example 9 – Late replating on different coatings
Shef-1 hESCs were seeded onto Matrigel coated T25 flask at a density of 240000 cells/cm2.
On Day 2 post seeding, 1μM LDN193189 and 10μM SB-431542 were applied for 4 days. On
10 Day 6, BMP4/7 was added to the media for 3 days. Cells were then maintained in media
alone until Day 50. On Day 50, the outer edge of the flask, where cobblestoned cells were
visible (Figure 8A), was collected and seeded onto Matrigel, Cellstart or Fibronectin coated
plates in 96-well or 48-well format at a density of 200000 cells/cm2. The inner dense area of
the flask, where cobblestones were not visible, was collected and seeded separately (Figure
15 8A). Replated cells were maintained in media alone or media supplemented with 0.5mM
cAMP. Cells replated from the inner dense area gave rise to a high proportion of neurons
and were discarded. Cells cultured from the outer edge gave rise to cobblestoned cells which
were more pigmented in the presence of cAMP (Figure 8B). Furthermore, cells expressed
RPE markers such as PMEL17, ZO-1, CRALBP, Bestrophin and MERTK as observed by
20 immunostaining. Quantification for PMEL17 and CRALBP immunostaining 15 days post
replating showed greater than 70% expression of both markers (Figure 8C). Similar
phenotypes were obtained on all coatings tested.
Example 10 – RPE cells obtained by Directed Differentiation closely resemble spontaneously
25 differentiated RPE cells
a) Preparation of spontaneously differentiated RPE cells
Shef-1 hESCs were cultured as colonies either on inactivated mouse embryonic fibroblasts
30 (iMEF) or inactivated human dermal fibroblasts (iHDFs) in Knockout DMEM (GIBCO)
supplemented with 20% KSR (GIBCO), 1% non-essential amino acid solution (GIBCO), 1
mM L-glutamine, 0.1 mM -mercaptoethanol, 30 g/ml gentamicin (GIBCO) and 4 ng/ml
human recombinant bFGF, or feeder free on Matrigel (BD) in mTesR1 medium (StemCell
Technologies). All cultures were fed daily until superconfluent (approximately 2 weeks post
35 seeding) before changing to Knockout DMEM media as above but without bFGF. Flasks
were fed thrice weekly until RPE colonies had appeared and were large enough to cut out.
The colonies were then excised with a scalpel, washed with PBS (-/-) and incubated with
37
Accutase (GIBCO) for 1-1.5 hrs in a shaking water bath. Dissociated RPE cells were
strained through a 70 m cell strainer, centrifuged at 700xg for 5min and resuspended in
warm Knockout DMEM media without bFGF as above. RPE cells were counted and seeded
(typically at 38000-50000 cells/cm2) into 48 well plates coated with extracellular matrix
(typically 1:50 CellStart (Life Technologies) in PBS (+/+) coated for 2 hrs in the cell cultur5 e
incubator). These were typically cultured for 7 or 16 weeks (cells seeded on day 0), feeding
twice weekly with 0.5 ml/well, before performing RNA extraction.
De-differentiated RPE cells samples were produced by the same protocol as above but cells
were seeded at 2500 cells/cm2 for de-differentiation and were cultured for 4 or 5 weeks.
10
b) Comparison of samples from RPE cells obtained by directed differentiation and
spontaneous differentiation
Samples obtained from directed differentiation as disclosed in Example 8 were compared
15 with samples obtained by spontaneous differentiation for a panel of RPE cells and other
markers by quantitative PCR. The spontaneously differentiated RPE cells had been in culture
for either 7 or 16 weeks. De-differentiated samples were used as a control as these cells did
not achieve an epithelial phenotype and instead remained fusiform and de-differentiated.
These were included to see whether the genes tested by qPCR were capable of
20 differentiating between epithelial RPE cells and non-RPE like cells.
Figure 9A shows a Principal Component Analysis (PCA) plot of 7 RPE cells samples
generated by directed differentiation along with RPE cells generated by spontaneous
differentiation as well as de-differentiated controls. Loadings plots of the PCA model of the
mean-centred, unit variance scaled mRNA transcript data are also shown which shows the
25 contribution of each of the genes tested to the clustering of the samples (Figure 9B). PCA
was used to visualise the overall variation of the samples. The scores plot of the first 2
components revealed that the de-differentiated samples clustered outside the Hotelling’s T2
ellipse and were characterised by lower levels of the markers positively correlated with the
RPE phenotype: MERTK, PMEL17, Tyrosinase, Bestrophin, RPE65 and CRALBP indicating
30 that they did not resemble differentiated RPE cells and that the genes tested were capable of
distinguishing between the RPE and non-RPE phenotype. Furthermore, RPE cells generated
by directed differentiation clustered with the RPE cells samples generated by spontaneous
differentiation and so possess the appropriate characteristics associated with differentiated
RPE cells.
35
Next, whole genome transcript profiling of RPE cells obtained by Directed Differentiation
(both Early and Late replating as disclosed in Examples 1 and 8) was performed and
38
compared with the transcript profile of RPE cells obtained by Spontaneous Differentiation.
The clustering of samples evident from the principal component analysis shown in Figure 9C
demonstrates that cells derived from both early and late replating protocols as disclosed in
Examples 1 and 8 have a genome-wide gene expression profile similar to RPE cells derived
from spontaneous differentiation, but distinct from hESCs5 .
In a related study, it was confirmed that RPE cells obtained by Spontaneous Differentiation
were similar to native RPE cells in terms of their gene expression signature.
10 Example 11 - RPE cells obtained by Directed Differentiation secrete VEGF and PEDF
proteins
a) RPE obtained by the early replating method
15 Cells obtained after Replate 2 (D9-19-50) of the early replating protocol disclosed in example
1 were seeded onto Transwells® at a density of 116000 cells/Transwell® and cultured for a
period of 10 weeks. The two chambers of the Transwell® were maintained as separate and
media were not allowed to mix. Media were collected from the bottom and top chamber and
analysed for secretion of VEGF and PEDF. As shown in Figure 10A, the ratio of
20 [VEGF]:[PEDF] is higher in the media collected from the bottom chamber and lower in the
media from the top chamber indicating higher basolateral secretion of VEGF and higher
apical secretion of PEDF. This indicates that the RPE obtained by directed differentiation
method disclosed herein are polarized and functional.
25 b) RPE obtained by the late replating method
For late replating, Shef-1 hESCs were seeded onto Matrigel coated T25 flask at a density of
240000 cells/cm2. On Day 2 post seeding, 1μM LDN193189 and 10μM SB-431542 were
applied for 4 days. On Day 6, 100ng/ml BMP4/7 was added to the media for 3 days. From
30 Day 9 onwards, cells were then maintained in media alone until Day 64 when outer edges of
the flask were collected and replated onto Matrigel coated Transwells® at a density of
400000 cells/cm2. The Transwell® were fed by overflowing twice a week. Spent media was
collected from Day 12 post seeding on Transwell® onwards at regular intervals for
quantification of VEGF and PEDF levels. VEGF and PEDF measurements were made using
35 the ‘Meso Scale Discover’ (MSD)-based multianalyte approach, according to the
manufacturer protocols. As shown on Figure 10B, VEGF and PEDF levels increase with time
39
in culture indicating active secretion by RPE cells, which is an indicator of maturity. These
results demonstrate that the cells obtained by the method described herein are RPEs.
Example 12 - Expansion of RPE cells
5
Proliferation of RPE cells is associated with a loss of the differentiated epithelial morphology
instead of which cells become elongated and fibroblastic in appearance. This apparent ‘dedifferentiation’
is followed by a phase of ‘re-differentiation’ where a confluent monolayer of
cells take up the characteristic phenotype of cuboidal-shaped, pigmented RPE cells (Vugler
10 et Al., Exp Neurol. 2008 Dec;214(2):347-61). This de-differentiation re-differentiation
paradigm, which occurs during expansion, has been described as an Epithelial-
Mesenchymal Transition (EMT) followed by a Mesenchymal-Epithelial Transition (MET)
(Tamiya et Al., IOVS, May 2010, Vol. 51, No. 5) (Figure 11 A).

We Claim:
1. A method for producing retinal pigment epithelial (RPE) cells comprising the steps of:
(a) culturing pluripotent cells in the presence of a first SMAD inhibitor and a second SMAD
inhibit5 or;
(b) culturing the cells of step (a) in the presence of a BMP pathway activator and in the
absence of the first and second SMAD inhibitors; and,
(c) replating the cells of step (b).
10 2. The method according to claim 1 wherein, in step (a), the cells are cultured as a
monolayer.
3. The method according to claim 1 or 2 wherein, in step (b), the cells are cultured as a
monolayer.
15
4. The method according to claim 1 wherein, in step (a), the cells are cultured in a
suspension culture.
5. The method according to any one of claims 1, 2 or 4 wherein, in step (b), the cells are
20 cultured in a suspension culture.
6. The method according to any one of claims 1 to 5, wherein the pluripotent cells are
selected from embryonic stem cells or induced pluripotent stem cells.
25 7. The method according to any one of claims 1 to 6, wherein the pluripotent cells are human
cells.
8. The method according to any one of claims 1 to 7, wherein the pluripotent cells are human
embryonic stem cells.
30
9. The method according to any one of claims 1 to 7, wherein the pluripotent cells are human
induced pluripotent stem cells.
10. The method according to any one of claims 1 to 9, wherein the pluripotent cells are
35 obtained by means which do not require the destruction of a human embryo.
54
11. The method according to any one of claims 1 to 10 wherein the first SMAD inhibitor is an
inhibitor of BMP type 1 receptor ALK2.
12. The method according to any one of claims 1 to 11 wherein the first SMAD inhibitor is an
inhibitor of BMP type 1 receptors ALK2 and ALK5 3.
13. The method according to any one of claims 1 to 12 wherein the first SMAD inhibitor
prevents Smad1, Smad5 and/or Smad8 phosphorylation.
10 14. The method according to any one of claims 1 to 13 wherein the first SMAD inhibitor is a
dorsomorphin derivative.
15. The method according to any one of claims 1 to 13 wherein the first SMAD inhibitor is
selected from dorsomorphin, noggin or chordin.
15
16. The method according to any one of claims 1 to 13 wherein the first SMAD inhibitor is 4-
(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (LDN193189) or a salt or
hydrate thereof.
20 17. The method according to any one of claims 1 to 16 wherein, in step (a), the concentration
of first SMAD inhibitor is between 0.5nM and 10μM.
18. The method according to any one of claims 1 to 17 wherein, in step (a), the concentration
of first SMAD inhibitor is between 500nM and 2μM.
25
19. The method according to any one of claims 1 to 18 wherein, in step (a), the concentration
of first SMAD inhibitor is about 1μM.
20. The method according to any one of claims 1 to 19 wherein the second SMAD inhibitor is
30 an inhibitor of ALK5.
21. The method according to any one of claims 1 to 20 wherein the second SMAD inhibitor is
an inhibitor of ALK5 and ALK4.
35 22. The method according to any one of claims 1 to 21 wherein the second SMAD inhibitor is
an inhibitor of ALK5 and ALK4 and ALK7.
55
23. The method according to any one of claims 1 to 20 wherein the second SMAD inhibitor is
selected from :
4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide;
2-methyl-5-(6-(m-tolyl)-1H-imidazo[1,2-a]imidazol-5-yl)-2H-benzo[d][1,2,3]triazole;
2-(6-methylpyridin-2-yl)-N-(pyridin-4-yl)quinazolin-4-amine5 ;
2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine;
4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)phenol;
2-(4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[3,2-c]pyridine;
4-(5-(3,4-dihydroxyphenyl)-1-(2-hydroxyphenyl)-1H-pyrazol-3-yl)benzamide;
10 2-(5-chloro-2-fluorophenyl)-N-(pyridin-4-yl)pteridin-4-amine;
6-methyl-2-phenylthieno[2,3-d]pyrimidin-4(3H)-one;
3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A 83-01);
2-(5-Benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine (SB-505124);
7-(2-morpholinoethoxy)-4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline
15 (LY2109761);
4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline (LY364947); or,
4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide (SB-431542)
or a salt or hydrate thereof.
20 24. The method according to any one of claims 1 to 20, wherein the second SMAD inhibitor
is 4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide (SB-431542).
25. The method according to any one of claims 1 to 24 wherein, in step (a), the concentration
of second SMAD inhibitor is between 0.5nM and 100μM.
25
26. The method according to any one of claims 1 to 25 wherein, in step (a), the concentration
of second SMAD inhibitor is between 1μM and 50μM.
27. The method according to any one of claims 1 to 26 wherein, in step (a), the concentration
30 of second SMAD inhibitor is about 10μM.
28. The method according to any one of claims 1 to 27 wherein, in step (a), the pluripotent
cells are cultured for at least 1 day.
35 29. The method according to any one of claims 1 to 28 wherein, in step (a), the pluripotent
cells are cultured for at least 2 days.
56
30. The method according to any one of claims 1 to 29 wherein, in step (a), the pluripotent
cells are cultured for between 2 and 10 days.
31. The method according to any one of claims 1 to 30 wherein, in step (a), the pluripotent
cells are cultured for between 3 and 5 day5 s.
32. The method according to any one of claims 1 to 31 wherein, in step (a), the pluripotent
cells are cultured for about 4 days.
10 33. The method according to any one of claims 1 to 32 wherein, before step (a), the cells are
cultured as a monolayer at an initial density of at least 1000 cells/cm2.
34. The method according to any one of claims 1 to 33 wherein, before step (a), the cells are
cultured as a monolayer at an initial density of between 100000 and 500000 cells/cm2.
15
35. The method according to any one of claims 1 to 34 wherein the BMP pathway activator
comprises a BMP.
36. The method according to any one of claims 1 to 35 wherein the BMP pathway activator
20 comprises a BMP selected from BMP2, BMP3, BMP4, BMP6, BMP7, BMP8, BMP9, BMP10,
BMP11 or BMP15.
37. The method according to any one of claims 1 to 36 wherein the BMP pathway activator is
a BMP homodimer.
25
38. The method according to any one of claims 1 to 36 wherein the BMP pathway activator is
a BMP heterodimer.
39. The method according to any one of claims 1 to 36 wherein the BMP pathway activator is
30 a BMP2/6 heterodimer, a BMP4/7 heterodimer or a BMP3/8 heterodimer.
40. The method according to any one of claims 1 to 36 wherein the BMP pathway activator is
a BMP4/7 heterodimer.
35 41. The method according to any one of claims 1 to 40 wherein, in step (b), the concentration
of BMP pathway activator is between 1ng/mL and 10μg/mL.
57
42. The method according to any one of claims 1 to 41 wherein, in step (b), the concentration
of BMP pathway activator is between 50 ng/mL and 500ng/mL.
43. The method according to any one of claims 1 to 42 wherein, in step (b), the concentration
of BMP pathway activator is about 100ng/5 mL.
44. The method according to any one of claims 1 to 43 wherein, in step (b), said cells are
cultured for at least 1 day.
10 45. The method according to any one of claims 1 to 44 wherein, in step (b), said cells are
cultured for between 2 days and 20 days.
46. The method according to any one of claims 1 to 45 wherein, in step (b), said cells are
cultured for about 3 days.
15
47. The method according to any one of claims 1 to 46 wherein, in step (c), said cells are
replated at a density of at least 1000 cells/cm2.
48. The method according to any one of claims 1 to 47 wherein, in step (c), said cells are
replated at a density of between 100000 and 1000000 cells/cm220 .
49. The method according to any one of claims 1 to 48 wherein, in step (c), said cells are
replated at a density of about 500000 cells/cm2.
25 50. The method according to any one of claims 1 to 49 wherein, in step (c), said cells are
replated on Matrigel®, fibronectin or Cellstart®.
51. The method according to any one of claims 1 to 50, wherein said method further
comprises the following steps:
30 (d) culturing the replated cells of step (c) in the presence of an activin pathway activator;
(e) replating the cells of step (d); and,
(f) culturing the replated cells of step (e).
52. The method according to claim 51 wherein, in step (d), the cells are cultured for at least 1
35 day.
58
53. The method according to claim 51 or 52 wherein, in step (d), the cells are cultured for at
least 3 days.
54. The method according to any one of claims 51 to 53 wherein, in step (d), the cells are
cultured for between 3 and 20 day5 s.
55. The method according to any one of claims 51 to 54 wherein, in step (d), the
concentration of activin pathway activator is between 1ng/mL and 10μg/mL.
10 56. The method according to any one of claims 51 to 55 wherein, in step (d), the
concentration of activin pathway activator is about 100ng/mL.
57. The method according to any one of claims 51 to 56 wherein, in step (d), the activin
pathway activator is activin A.
15
58. The method according to any one of claims 51 to 57 wherein, in step (d), the cells are
cultured in the presence of cAMP.
59. The method according to claim 58 wherein, in step (d), the concentration of cAMP is
20 about 0.5mM.
60. The method according to any one of claims 51 to 59 wherein, in step (e), the cells are
replated at a density of at least 1000 cells/cm2.
25 61. The method according to any one of claims 51 to 60 wherein, in step (e), said cells are
replated at a density of between 20000 and 500000 cells/cm2.
62. The method according to any one of claims 51 to 61 wherein, in step (e), said cells are
replated at a density of about 200000 cells/cm2.
30
63. The method according to any one of claims 51 to 62 wherein, in step (e), said cells are
replated on Matrigel®, fibronectin or Cellstart®.
64. The method according to any one of claims 51 to 63 wherein, in step (f), the cells are
35 cultured for at least 5 days.
59
65. The method according to any one of claims 51 to 64 wherein, in step (f), the cells are
cultured for at least 14 days.
66. The method according to any one of claims 51 to 65 wherein, in step (f), the cells are
cultured for between 10 and 35 day5 s.
67. The method according to any one of claims 51 to 66 wherein, in step (f), the cells are
cultured for about 28 days.
10 68. The method according to any one of claims 51 to 67 wherein, in step (f), the cells are
cultured in the presence of cAMP.
69. The method according to claim 68 wherein, in step (f), the concentration of cAMP is
about 0.5mM.
15
70. The method according to any one of claims 1 to 50, wherein,
step (b) further comprises, after culturing the cells in the presence of the BMP pathway
activator, culturing the cells for at least 10 days in the absence of the BMP pathway activator;
step (c) comprises replating the cells of step (b) having a cobblestone morphology; and said
20 method further comprising the step of:
(d) culturing the replated cells of step (c).
71. The method according to claim 70 wherein, in step (b), the cells are cultured for at least
20 days in the absence of BMP pathway activator.
25
72. The method according to claim 70 or 71 wherein, in step (b), the cells are cultured for
between 30 and 50 days in the absence of BMP pathway activator.
73. The method according to any one of claim 70 to 72 wherein, in step (b), the cells are
30 cultured for about 40 days in the absence of BMP pathway activator.
74. The method according to any one of claims 70 to 73 wherein, in step (c), the cells are
replated at a density of at least 1000 cells/cm2.
35 75. The method according to any one of claims 70 to 74 wherein, in step (c), the cells are
replated at a density of between 50000 and 500000 cells/cm2.
60
76. The method according to any one of claims 70 to 75 wherein, in step (c), the cells are
replated at a density of about 200000 cells/cm2.
77. The method according to any one of claims 70 to 76 wherein, in step (c), said cells are
replated on Matrigel®, fibronectin or Cellstart5 ®.
78. The method according to anyone of claims 70 to 77 wherein, in step (d), the cells are
cultured for at least 5 days.
10 79. The method according to anyone of claims 70 to 78 wherein, in step (d), the cells are
cultured for between 10 and 40 days.
80. The method according to anyone of claims 70 to 79 wherein, in step (d), the cells are
cultured for about 14 days.
15
81. The method according to any one of claims 70 to 80 wherein, in step (d), the cells are
cultured in the presence of cAMP.
82. The method according to claim 81 wherein, in step (d), the concentration of cAMP is
20 about 0.5mM.
83. The method according to any one of claims 70 to 82 comprising the following additional
steps:
(e) replating the cells of step (d);
25 (f) culturing the replated cells of step (e).
84. The method according to claim 83 wherein, in step (e), the cells are replated at a density
of at least 1000 cells/cm2.
30 85. The method according to claim 83 or 84 wherein, in step (e), the cells are replated at a
density of between 50000 and 500000 cells/cm2.
86. The method according to any one of claims 83 to 85 wherein, in step (e), the cells are
replated at a density of about 200000 cells/cm2.
35
87. The method according to any one of claims 70 to 86, wherein, in step (e), said cells are
replated on Matrigel®, fibronectin or Cellstart®.
61
88. The method according to anyone of claims 70 to 87 wherein, in step (f), the cells are
cultured for at least 10 days.
89. The method according to anyone of claims 70 to 88 wherein, in step (f), the cells ar5 e
cultured for between 15 and 40 days.
90. The method according to anyone of claims 70 to 89 wherein, in step (f), the cells are
cultured for about 28 days.
10
91. The method according to any one of claims 1 to 90 wherein said method further
comprises the step of harvesting the RPE cells.
92. The method according to any one of claims 1 to 91 wherein said method further
15 comprises the step of purifying the RPE cells.
93. The method according to any one of claims 1 to 91 wherein said method further
comprises the step of purifying the RPE cells by Fluorescence Activated Cell Sorting (FACS)
or Magnetic Activated Cell Sorting (MACS).
20
94. The method according to claim 92 wherein said step of purifying the RPE cells comprises
the step of:
- contacting the cells with an anti-CD59 antibody conjugated to a fluorophore, and,
- selecting the cells that bind to the anti-CD59 antibody using FACS.
25
95. The method according to claim 92 wherein said step of purifying the RPE cells comprises
the step of:
- contacting the cells with an anti-CD59 antibody conjugated to a magnetic particle, and,
- selecting the cells that bind to the anti-CD59 antibody using MACS.
30
96. The method according to any one of claims 1 to 90 wherein, in all steps, the cells are
cultured as a monolayer.
97. The method according to any one of claims 1 to 96 wherein the RPE cells are expanded
35 by a method comprising
- replating RPE cells; and,
- culturing the replated RPE cells.
62
98. The method according to claim 97 wherein the cells are replated at a density between
1000 and 100000 cells/cm2.
99. The method according to claim 97 or 98 wherein the cells are replated at a densit5 y
between 10000 and 30000 cells/cm2.
100. The method according to any one of claims 97 to 99 wherein the cells are replated at a
density of about 20000 cells/cm2.
10
101. The method according to any one of claims 97 to 100 wherein the cells are replated on
Matrigel®, Fibronectin or Cellstart®.
102. The method according to any one of claims 97 to 101, wherein the cells are cultured for
15 at least 7 days, at least 14 days, at least 28 days or at least 42 days.
103. The method according to any one of claims 97 to 102, wherein the cells are cultured for
about 49 days.
20 104. The method according to any one of claims 97 to 103, wherein the cells are cultured in
the presence of a SMAD inhibitor, cAMP or an agent which increases the intracellular
concentration of cAMP.
105. The method according to claim 104, wherein said agent is selected from an Adenyl
25 Cyclase activator, preferably forskolin or a phosphodiesterase (PDE) inhibitor, preferably a
PDE1, PDE2, PDE3, PDE4, PDE7, PDE8, PDE10 and/or PDE11 inhibitor.
106. The method according to claim 104 or 105, wherein said the cells are cultured in the
presence of cAMP.
30
107. The method according to claim 106, wherein the concentration of cAMP is between
0.01mM and 1M.
108. The method according to claim 106 or 107, wherein the concentration of cAMP is about
35 0.5mM.
109. A method for expanding RPE cells comprising the following steps:
63
(a) plating RPE cells at a density of at least 1000 cells/cm2, and,
(b) culturing said RPE cells in the presence of SMAD inhibitor, cAMP or an agent which
increases the intracellular concentration of cAMP.
110. The method according to claim 109, wherein, in step (a), the cells are plated at 5 a
density between 5000 and 100000 cells/cm2.
111. The method according to claim 109 or 110, wherein, in step (a), the cells are plated at a
density about 20000 cells/cm2.
10
112. The method according to any one of claims 109 to 111, wherein, in step (a), the cells
are plated on Matrigel®, Fibronectin or Cellstart®.
113. The method according to any one of claims 109 to 112, wherein, in step (b), the cells
15 are cultured for at least 7 days, at least 14 days, at least 28 days or at least 42 days.
114. The method according to any one of claims 109 to 113, wherein, in step (b), the cells
are cultured for about 49 days.
20 115. The method according to any one of claims 109 to 114, wherein said agent is selected
from an adenyl Cyclase activator, preferably forskolin or a phosphodiesterase (PDE)
inhibitor, preferably a PDE1, PDE2, PDE3, PDE4, PDE7, PDE8, PDE10 and/or PDE11
inhibitor.
25 116. The method according to any one of claims 109 to 114, wherein, in step (b), the cells
are cultured in the presence of cAMP.
117. The method according to claim 116, wherein the concentration of cAMP is between
0.01mM and 1M.
30
118. The method according to claim 116 or 117, wherein the concentration of cAMP is about
0.5mM.
119. The method according to any one of claims 109 to 114, wherein, in step (b), the cells
35 are cultured in the presence of a SMAD inhibitor.
64
120. The method according to claim 119, wherein the SMAD inhibitor is 2-(6-methylpyridin-2-
yl)-N-(pyridin-4-yl)quinazolin-4-amine, 6-(1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)quinazolin-
4(3H)-one, or 4-methoxy-6-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)quinoline.
121. The method according to any one of claims 1 to 120 wherein the produced RPE cell5 s
have a cobblestone morphology, are pigmented and express at least one of the following
RPE markers: MITF, PMEL17, CRALBP, MERTK, BEST1 and ZO-1.
122. The method according to any one of claims 1 to 121 wherein the produced RPE cells
10 secrete VEGF and PEDF.
123. The method according to any one of claims 1 to 122 wherein all steps are carried out in
xeno-free conditions.
15 124. RPE cells obtained by a method according to anyone of claims 1 to 123.
125. RPE cells obtainable by a method according to anyone of claims 1 to 123.
126. A pharmaceutical composition comprising the RPE cells of claim 124 or 125.
20
127. A method for the treatment of a retinal disease in a subject, said method comprising
administering RPE cells of claim 124 or 125 or a pharmaceutical composition of claim 126 to
said subject.
25 128. A method for producing RPE cells comprising:
a) providing a population of pluripotent cells;
b) inducing the differentiation of pluripotent cells into RPE cells, and,
c) enriching the cell population for cells expressing CD59.
30 129. The method according to claim 128 wherein step c) comprises
- contacting the cells with an anti-CD59 antibody conjugated to a fluorophore, and,
- selecting the cells that bind to the anti-CD59 antibody using FACS.
130. The method according to claim 128 wherein step c) comprises
35 - contacting the cells with an anti-CD59 antibody conjugated to a magnetic particle, and,
- selecting the cells that bind to the anti-CD59 antibody using MACS.
65
131. A method for purifying RPE cells comprising:
a) providing a cell population comprising RPE cells and non RPE cells;
b) increasing the percentage of RPE cells in the cell population by enriching the cell
population for cells expressing CD59.
5
132. The method according to claim 131 wherein step b) comprises
- contacting the cell population with an anti-CD59 antibody conjugated to a fluorophore, and,
- selecting the cells that bind to the anti-CD59 antibody using FACS.
10 133. The method according to claim 131 wherein step b) comprises
- contacting the cell population with an anti-CD59 antibody conjugated to a magnetic particle,
and,
- selecting the cells that bind to the anti-CD59 antibody using MACS.
15 134. The method according to anyone of claims 131 to 133 wherein the non RPE cells are
pluripotent cells or RPE progenitors.