WO2010029288A2

WO2010029288A2 - Growth factor

Info

Publication number: WO2010029288A2
Application number: PCT/GB2009/002142
Authority: WO
Inventors: James Walsh
Original assignee: Axordia Limited
Priority date: 2008-09-11
Filing date: 2009-09-08
Publication date: 2010-03-18
Also published as: WO2010029288A3; GB0816577D0

Abstract

This disclosure relates to cell lines and uses of cell lines that express Wingless [Wnt] or homologues thereof; methods to amplify retinal progenitor cells; and differentiation of said progenitor cells into retinal pigment epithelial cells.

Description

Growth Factor

The invention relates to cell lines and uses of cell lines that express Wingless in particular [Wnt] 2B or homologues thereof; methods to amplify retinal progenitor cells; and differentiation of said progenitor cells into retinal pigment epithelial cells.

The ability to see is dependent on the actions of several structures in and around the eye. When one focuses on an object, light rays are reflected from the object to the cornea. The light rays are bent, refracted and focused by the cornea, lens, and vitreous within the eye ball. ThQ lens functions to ensure that light is focused on the retina. The retina then converts light into electrical impulses which are transmitted through the optic nerve to the brain where the image is perceived. The retina is comprised of several cell types arranged in layers. The photoreceptor cells [rods and cones] are supported on a specialized pigmented epithelial layer referred to as the retinal pigment epithelium [RPE].

The RPE is derived from neuroectoderm and is essential for maintenance of the photoreceptor cells. The RPE is located between the choroid and neural retina and functions in part as a barrier between the blood stream and retina. Its functions include phagocytosis of shed rod and cone outer segments, absorption of stray light, vitamin A metabolism, regeneration of retinoids and tissue repair. Differentiated RPE cells have known markers and these include cellular retinaldehyde-binding protein, RPE65, a cytoplasmic protein involved in retinoid metabolism, bestrophin which is the product of the Best vitelliform macular dystrophy gene [VMD2] and pigment epithelium derived factor [PEDF]. RPE cells are normally mitotically quiescent but can initiate division in response to injury. RPE cells near to an injury flatten and proliferate to form a new epithelial monolayer and have the ability to produce fibroblast like cells which can differentiate into RPE cells. In mammalian development the RPE has the same progenitor as neural retina which is the neuro-epithelium of the optic vesicle.

Macular degeneration is an age related condition that results from atrophy in the macula area of the retina. This can result in loss of central vision and is a leading cause of blindness in those over the age of 50. People with early stage age-related macular degeneration [AMD], also called age related maculopathy, do not necessarily have poor vision and have characteristic yellow deposits in the macula. Advanced AMD results in significant loss of vision and can be of two types, dry and wet. Dry AMD results in atrophy of the RPE and consequent loss of photoreceptors in the central macula. There is no treatment for this condition although there is evidence that dietary supplements [e.g. lutein, zeaxanthin] can slow the progress of the disease. Wet AMD, also called neovascular or exudative AMD results from abnormal blood capillary growth which results in leakage of blood and protein below the macula. This results in scarring and irreversible damage to photoreceptors. The treatment of wet AMD involves the use of anti-angiogenics which are directly injected into the eye to inhibit new capillary formation. In addition laser treatment to remove capillary growth is common altjough has undesirable side effects. The degeneration of the RPE is thought to play a critical role in AMD. Animal studies have been conducted that indicate that cell based therapies may be successful in treating diseases of the retina. The transplantation of RPE cells into the retina of an animal model of AMD has resulted in the rescue of host photoreceptors and an attenuation of vision [see Coffey et al 2002 Nat Neuroscience. 5. 53-56; Lund et al 2001 Proc. Natl. Acad. Sci USA, 98, 9942-9947].

Wnt genes encode diffusible, extracellular signalling molecules of around 350-400 amino acids in length, defined by a characteristic pattern of conserved cysteine residues, along with other invariant amino acids. The Wnt family is composed of more than 60 members, with 14 human homologues alone. Well-documented roles exist for Wnt signalling in a variety of developmental processes, including cell fate specification and patterning within the central nervous system. Wnt ligands interact with membrane-bound receptors of the frizzled family, leading to activation of a cytoplasmic protein, Dishevelled. Wnt signalling may be antagonised by extracellular molecules that compete for Wnt binding, including frizzled related proteins (FRP), Wnt inhibitory factors (WIF), Dickkopf (DKK) and Cerberus (CER).

According to an aspect of the invention there is provided a method to enhance the proliferation and/or differentiation of progenitor retinal pigmented epithelial cells comprising: i) providing a cell culture preparation comprising: progenitor retinal pigmented epithelial cells, a cell culture surface and a cell culture medium wherein said medium is supplemented with a polypeptide that has the activity associated with a Wnt polypeptide; and i) providing cell culture conditions that enhance the proliferation and/or differentiation of progenitor retinal pigmented epithelial cells. In a preferred method of the invention said Wnt polypeptide is selected from the group consisting of: WNT 1 , WNT 2, WNT 2b variant 1 , WNT 2b variant 2, WNT 3, WNT 3A, WNT 4, WNT 5A, WNT 5B-1 , WNT 5B-2, WNT 6, WNT 7A, WNT 7B₁ WNT 8A, WNT 8B, WNT 9A, WNT 9B, WNT 10A, WNT 1OB WNT 11 , WNT 16 or WNT 16 variant 2.

In a further preferred method of the invention said polypeptide is WNT 2b variant 1 or WNT 2b variant 2.

In a further preferred method of the invention said polypeptide is selected from the group represented by the amino acid sequences in Figures 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a, 17a, 18a, 19a, 20a, 21a or 22a or polypeptide variants thereof.

Polypeptide variants are polypeptide sequences having at least 75% identity with the polypeptide sequences as herein disclosed, or fragments and functionally equivalent polypeptides thereof. In one embodiment, the polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the amino acid sequences illustrated herein. Preferably said Wnt polypeptide is human.

In a preferred method of the invention said Wnt polypeptide is exogenously added to said culture medium.

In an alternative preferred method of the invention said cell culture medium comprises a cell line that produces said Wnt polypeptide.

In a preferred method of the invention said cell line naturally produces said Wnt polypeptide.

In an alternative preferred method of the invention said cell line is transfected with a nucleic acid molecule that encodes said Wnt polypeptide. In a preferred method of the invention said transfected nucleic acid molecule is operably linked to a promoter that regulates the expression of said nucleic acid molecule.

In a preferred method of the invention said cell culture medium further comprises at least one Wnt polypeptide antagonist to inhibit Wnt signalling thereby, either directly or indirectly inducing differentiation of progenitor retinal pigmented epithelial cells.

In a preferred method of the invention said Wnt polypeptide antagonist is an antibody.

In an alternative preferred method of the invention said Wnt polypeptide antagonist is a natural inhibitor of Wnt signalling.

Preferably said natural Wnt polypeptide inhibitor is selected from the group consisting of: CER1 , WIF 1 , SFRP1 , SFRP2, SFRP4, SFRP5, FRZB, DKK1, DKK2, DKK3 (transcript variants 1-3), DKK4 or DKKL1.

In a preferred method of the invention said Wnt polypeptide inhibitor is represented by the amino acid sequence in Figure 23a, 24a, 25a, 26a, 27a, 28a, 29a, 30a, 31a, 32a, 33a, 34a, 35a or 36a, or functional variant thereof.

According to a further aspect of the invention there is provided a cell culture comprising: progenitor retinal pigmented epithelial cells and a cell culture medium wherein said medium is supplemented with a polypeptide that has the activity associated with a Wnt polypeptide.

In a preferred embodiment of the invention said cell culture medium comprises a polypeptide selected from the group consisting of: WNT 1, WNT 2, WNT 2b variant 1, WNT 2b variant 2, WNT 3, WNT 4, WNT 5A, WNT 5B, WNT 6, WNT 7A₁ WNT 7B, WNT 8A, WNT 8B, WNT 9A, WNT 9B, WNT 10A, WNT 10B WNT 11, WNT 16 or WNT 16 variant 2.

In a further preferred embodiment of the invention said polypeptide is WNT 2b variant 1 or WNT 2b variant 2.

In a further preferred embodiment of the invention said polypeptide is selected from the group represented by the amino acid sequences in Figures 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a, 17a, 18a, 19a, 20a, 21a or 22a, or polypeptide variants thereof.

In a preferred embodiment of the invention said cell culture medium comprises at least one Wnt polypeptide antagonist to inhibit Wnt signalling thereby, either directly or indirectly, inducing differentiation of progenitor retinal pigmented epithelial cells.

In a preferred embodiment of the invention said Wnt polypeptide antagonist is an antibody.

In an alternative preferred embodiment of the invention said Wnt polypeptide antagonist is a natural inhibitor of Wnt signalling.

In a preferred embodiment of the invention said Wnt polypeptide inhibitor is selected from the group consisting of: CER1 , WIF 1, SFRP1, SFRP2, SFRP4, SFRP5, FRZB, DKK1 , DKK2, DKK3 (transcript variants 1-3), DKK4 or DKKL1.

In a preferred embodiment of the invention said Wnt polypeptide inhibitor is represented by the amino acid sequence in Figure 23a, 24a, 25a, 26a, 27a, 28a 29a, 30a, 31a, 32a, 33a, 34a, 35a or 36a, or functional variant thereof.

According to an aspect of the invention there is provided the use of Wingless [Wnt] polypeptide in the stimulation of proliferation and/or differentiation of progenitor retinal pigmented epithelial cells [PRPE].

According to a further aspect of the invention there is provided cell line transfected with a nucleic acid molecule selected from the grqup consisting of: i) a nucleic acid molecule comprising or consisting of a nucleic acid sequence as represented by the sequences presented in Figures 1b, 2b,

3b, 4b, 5b, 6b, 7b, 8a, 9b, 10b, 11b, 12b, 13b, 14b, 15b, 16b, 17b, 18b, 19b, 20b, 21b or 22b; ii) a nucleic acid molecule comprising a sequence that hybridizes under stringent hybridization conditions to the molecules in i) above and encodes a polypeptide with the activity associated with a Wnt polypeptide, wherein said nucleic acid molecule is operably linked to a control sequence that regulates the expression of said nucleic acid molecule.

Typically, "operably linked" includes, by example and not by way of limitation, the provision of transcription control sequences (promoter sequences) which mediate cell/tissue specific expression. These promoter sequences may be cell/tissue specific, inducible or constitutive.

Promoter is an art recognised term and, for the sake of clarity, includes the following features which are provided by example only, and not by way of limitation. Enhancer elements are cis acting nucleic acid sequences often found 5' to the transcription initiation site of a gene (enhancers can also be found 3' to a gene sequence or even located in intronic sequences and is therefore position independent). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors (polypeptides) which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S

Latchman, Academic Press Ltd, San Diego) is responsive to a number of environmental cues. Promoter elements also include so called TATA box and RNA polymerase initiation selection (RIS) sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.

Adaptations also include the provision of selectable markers and autonomous replication sequences which both facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors. Episomal vectors are desirable since these molecules can incorporate large DNA fragments (30-50kb DNA). Episomal vectors of this type are described in WO98/07876. Adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bicistronic or multi-cistronic expression cassettes.

These adaptations are well known in the art. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach VoI III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, lnc.(1994).

In a preferred embodiment of the invention said cell line is transfected with a nucleic acid molecule comprising of a nucleic acid sequence as represented in Figure 3b or 4b.

According to an aspect of the invention there is provided the use of a cell according to the invention in the stimulation of proliferation and/or differentiation of progenitor retinal pigmented epithelial cells [PRPE].

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule that encodes a polypeptide with the activity associated with Wnt 2b; preferably said nucleic acid molecule comprises or consists of the nucleic acid sequence represented in Figures 3b or 4b.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures: Figure 1a is the amino acid sequence of human Wnt 1; Figure 1b is the nucleic acid sequence of Wnt 1;

Figure 2a is the amino acid sequence of human Wnt 2; Figure 2b is the nucleic acid sequence of Wnt 2;

Figure 3a is the amino acid sequence of human Wnt 2b variant 1; Figure 3b is the nucleic acid sequence of Wnt 2b variant 1 ;

Figure 4a is the amino acid sequence of human Wnt 2b variant 2; Figure 4b is the nucleic acid sequence of Wnt 2b variant 2;

Figure 5a is the amino acid sequence of human Wnt 3; Figure 5b is the nucleic acid sequence of Wnt 3;

Figure 6a is the amino acid sequence of human Wnt 3a; Figure 6b is the nucleic acid sequence of Wnt 3a;

Figure 7a is the amino acid sequence of human Wnt 4; Figure 7b is the nucleic acid sequence of Wnt 4;

Figure 8a is the amino acid sequence of human Wnt 5a; Figure 8b is the nucleic acid sequence of Wnt 5a;

Figure 9a is the amino acid sequence of human Wnt 5b-1 ; Figure 9b is the nucleic acid sequence of Wnt 5b-1 ;

Figure 10a is the amino acid sequence of human Wnt 5b-2: Figure 10b is the nucleic acid sequence of Wnt 5b-2;

Figure 11a is the amino acid sequence of human Wnt 6; Figure 11b is the nucleic acid sequence of Wnt 6

Figure 12a is the amino acid sequence of human Wnt 7a; Figure 12b is the nucleic acid sequence of Wnt 7a; Figure 13a is the amino acid sequence of human Wnt 7b; Figure 13b is the nucleic acid sequence of Wnt 7b

Figure 14a is the amino acid sequence of human Wnt 8a; Figure 14b is the nucleic acid sequence of Wnt 8a;

Figure 15a is the amino acid sequence of human Wnt 8b; Figure 15b is the nucleic acid sequence of Wnt 8b;

Figure 16a is the amino acid sequence of human Wnt 9a; Figure 16b is the nucleic acid sequence of Wnt 9a;

Figure 17a is the amino acid sequence of human Wnt 9b; Figure 17b is the nucleic acid sequence of Wnt 9b;

Figure 18a is the amino acid sequence of human Wnt 10a; Figure 18b is the nucleic acid sequence of Wnt 10a;

Figure 19a is the amino acid sequence of human Wnt 10b; Figure 19b is the nucleic acid sequence of Wnt 10b;

Figure 20a is the amino acid sequence of human Wnt 11 ; Figure 20b is the nucleic acid sequence of Wnt 11 ;

Figure 21a is the amino acid sequence of human Wnt 16; Figure 21b is the nucleic acid sequence of Wnt 16;

Figure 22a is the amino acid sequence of human Wnt 16 variant 2; Figure 22b is the nucleic acid sequence of Wnt 16 variant 2;

Figure 23a is the amino acid sequence of Homo sapiens Cerberus 1, cysteine knot superfamily, homolog (Xenopus laevis) (CER1); Figure 23b is the nucleic acid sequence;

Figure 24a is the amino acid sequence of Homo sapiens WNT inhibitory factor 1 (WIF1); Figure 24b is the nucleic acid sequence; Figure 25a is the amino acid sequence of Homo sapiens secreted frizzled-related protein 1 (SFRP1); Figure 25b is the nucleic acid sequence;

Figure 26a is the amino acid sequence of Homo sapiens secreted frizzled-related protein 2 (SFRP2); Figure 26b is the nucleic acid sequence;

Figure 27a is the amino acid sequence of Homo sapiens frizzled-related protein (FRZB); Figure 27b is the nucleic acid sequence;

Figure 28a is the amino acid sequence of Homo sapiens secreted frizzled-related protein

4 (SFRP4); Figure 28b is the nucleic acid sequence;

Figure 29a is the amino acid sequence of Homo sapiens secreted frizzled-related protein

5 (SFRP5); Figure 29b is the nucleic acid sequence;

Figure 30a is the amino acid sequence of Homo sapiens Dkk 1 polypeptide; Figure 30b is the nucleic acid sequence;

Figure 31a is the amino acid sequence of Homo sapiens Dkk 2 polypeptide; Figure 31b is the nucleic acid sequence;

Figure 32a is the amino acid sequence of Homo sapiens Dkk 3 variant 1 polypeptide; Figure 32b is the nucleic acid sequence;

Figure 33a is the amino acid sequence of Homo sapiens Dkk 3 variant 2 polypeptide; Figure 33b is the nucleic acid sequence;

Figure 34a is the amino acid sequence of Homo sapiens Dkk 3 variant 3 polypeptide; Figure 34b is the nucleic add sequence;

Figure 35a is the amino acid sequence of Homo sapiens Dkk 4 polypeptide; Figure 35b is the nucleic acid sequence;

Figure 36a is the amino acid sequence of Homo sapiens Dkk L1 polypeptide; Figure 36b is the nucleic acid sequence; B2009/002142

11

Figure 37 is the nucleic acid sequence of the human PGK promoter;

Figure 38 is the nucleic acid sequence of the human Ubiquitin C promoter;

Figure 39 is the nucleic acid sequence of the human EF-1 alpha promoter,

Figure 40 is the nucleic acid sequence of the human CMV promoter;

Figure 41 is the nucleic acid sequence of the human CAG promoter;

Figure 42 is the an IRS nucleic acid sequence;

Figure 43 is the nucleic acid sequence of pTRE-Tϊght;

Figure 44 illustrates a vector map of pTet-On-Advanced; and

Figure 45 illustrates western blot detection of Wnt 2b; 30, 20, 10 and 5 microlitre aliquots of 10x concentrated medium taken from a 577MF cell culture after two days exposure were run on a PAGE gel, blotted and probed for Wnt2b using a rabbit polyclonal antibody and anti-rabbit HRP conjugated secondary antibody for detection. Bands of decreasing intensity can be seen running from left to right, corresponding to the sample volume loading.

MATERIALS AND REAGENTS FOR CELL CULTURE

Tissue culture vessels:

T25 25cm2 plasma treated flasks: nunc/ThermoFisher Scientific cat #156340 T75 75cm2 plasma treated flasks: nunc/Thermo Fisher Scientific cat #156472

Preparation of human embryonic stem cell (HES) medium: Add together:

160ml Knockout DMEM

40ml Knockout SR 2ml solution 1*

2ml 100x non essential amino acids 400μl of a 2μg/μl bFGF stock Antibiotics (if required)

*Solution 1: Add 10mI PBS (-Ca²⁺, Mg²⁺) to 0.146g L-glutamine

Add 7μl β-metcaptoethanol to the PBS/L-Glutamine solution and mix well

Formulation of HES medium and component sources:

Final concentration Manufacturer cat. number

80% Knockout DMEM Gibco 10829-018

20% GIBCO Knockout SR Gibco 10828-028

1 % Non essential amino acids Gibco 11140-035

1mM L-Glutamine Gibco 21051-016 0.1μM β -mercaptoethanol Sigma M-7154

4ng/m! human bFGF Gibco 13256-029

Preparation of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS)

1. Heat inactivate the FBS by incubating in a water bath at 60 degrees C for 40 minutes (store at 4 degrees C afterwards)

2. Add 50ml of FBS to a 500ml bottle of DMEM

3. Mix thoroughly

Component sources:

DMEM (high glucose with L-glutamine) PAA Laboratories GmbH cat# E15-810

Foetal bovine serum (qualified) Invitrogen cat# 10099-141

Preparation of mitomycin C solution

Add 2mg of mitomycin C to 200ml of DMEM+10%FBS Component sources: Mitomycin C Sigma cat# M-4287 Preparation of trypsin solution Add 0.25% v/v Trypsin and EDTA to 5mM to PBS Component sources: Trypsin Gibco cat# 15090-046

EDTA Sigma cat# E-5134

USE OF WNT2B CONTAINING MEDIUM TO INCREASE THE YIELD OF PRECURSOR RPE CELLS AND RPE CELLS FROM HUMAN EMBRYONIC STEM CELLS

1. Aspirate and discard the medium from 5 day post-passage human embryonic stem cells in a T25 flask 2. Add 10 mis of Wnt2b containing medium to the flask

3. Place in an incubator at 37 degrees C/10% CO2

4. Repeat steps 1. to 3. every other day up to and including day 13 post passage

5. Aspirate and discard the medium from the flask at day 15 post passage

6. Add 10 mis of DMEM+10% FBS to the flask 7. Place the flask in an incubator at 37 degrees C/10% CO2

8. Feed as required, indicated by yellowing of the phenol red indicator in the medium, by aspirating and replacing the spent medium

9. Harvest or maintain pigmented colonies of RPE (typically appearing from day 19 post passage)

HUMAN EMBRYONIC STEM CELL CULTURE

Human embryonic stem cells can be grown in flasks with or without feeder cells; for preparation of culture vessels, follow A or B, respectively.

A: Fibroblast feeder coated tissue culture flask preparation

1. Add 5ml of 0.1% gelatine in PBS to each fresh T25 flasks to be used and leave at room temperature for at least 3 hours (can be stored at 4 degrees C for up to 7 days after preparation) 2. Aspirate and discard the medium from a culture of dividing embryonic fibroblasts in a T75 flask

3. Add sufficent mitomycin-C solution to cover the cells

4. Return the flask to the incubator at 37 degrees C/10% CO2 for 3 hours

5. Aspirate and discard the mitomycin-C solution 6. Add 5ml of DMEM+10% FBS to the flask

7. Gently agitate the flask (left-right and front-back rocking)

8. Aspirate and discard the medium 9. Add 1 ml of trypsin solution to the flask

10. Incubate at room temperature for 5 minutes

11. Vigorously agitate the flask for 10 seconds (jarring blows)

12. Add 10ml of DMEM+10% FBS to the flask 13. Gently pipette the cell suspension up and down to break up cell clumps and evenly distribute the cells

14. Count the cells to determine density of suspension

15. Seed the cells into the flasks prepared in step 1. at 6000 per cm2 in DMEM+10% FBS 16. Place in an incubator at 37 degrees C/10% CO2 for =/>24 hours

17. Use within 7 days

B: Feeder-free tissue culture flask preparation

1. Add 5ml of coating solution (e.g. Matrigel in DMEM at 1:15 dilution) to each fresh

T25 flask to be used

2. Incubate at room temperature for 2 hours or >24hr at 4 degC (can be stored at 4 deg C for up to 7 days)

3. Aspirate the coating solution immediately prior to use

PREPARATION OF FEEDER-CONDITIONED MEDIUM FOR GROWTH OF HUMAN EMBRYONIC STEM CELLS IN THE ABSENCE OF FEEDER CELLS

Follow method A: Fibroblast feeder coated tissue culture flask preparation, up to step 14, then:

15. Seed the cells at 56,000 cells per cm2 in DMEM+10% FBS

16. Place in an incubator at 37 degrees C/10% CO2

17. At least 6 hours after seeding, aspirate and discard the medium 18. Add fresh HES medium to a volume of 0.5cm3 per 1 cm2 of culture surface area

19. Place the flask in an incubator at 37 degrees C/5% CO2

20. Collect the feeder-conditioned HES medium after 24 hours

21. Replace the medium on the feeders with further, fresh HES medium and return to step 18 (this can be repeated up to 6 times for repeated harvests of feeder conditioned medium from a single flask) PASSAGE PROTOCOL FOR HUMAN EMBRYONIC STEM CELLS IN T25 TISSUE CULTURE FLASKS

1. Aspirate and discard the spent medium from a culture of human embryonic stem cells

2. Add 1 ml of collagenase Type IV solution (1 mg/ml in DMEM) to the flask

3. Incubate at 37degrees C for 10 minutes 4. Add approximately 20 glass beads (ready prepared in sterile vials) to the flask

5. Agitate the flask gently (horizontal shaking and rocking) for 30 seconds to dislodge the colonies of cells

6. Pipette the cell suspension repeatedly to fragment colonies into multicellular clumps (requires optimisation, as clump size is critical to successful passage) 7. Add 5ml of fresh HES medium equilibrated to 37 degrees C/5% CO2 into each new T25 flask to be used

8. Seed the cell suspension from 6. at a 1 :3 ratio into the flasks prepared in 7.

9. Agitate the flasks gently, with caps uppermost, to evenly distribute cells throughout suspension 10. Slowly lower the flasks to horizontal position

11. Place in an incubator and maintain at 37 degrees C/5% CO2

FEEDING PROTOCOL FOR HUMAN EMBRYONIC STEM CELLS IN T25 TISSUE CULTURE FLASKS

1. Aspirate and discard the spent medium from the flask

2. Add 5ml of fresh HES medium equilibrated to 37 degrees C/5% CO2

3. Return the flask to an incubator at 37 degC/5% CO2

ENDOGENOUS WNT2B EXPRESSION BY 577MF CELLS: PRODUCTION OF CONDITIONED MEDIUM CONTAINING WNT2B

MAINTENANCE AND PASSAGE OF CELLS 1. Add 1 ml of trypsin solution to a confluent T75 flask of 577MF cells

2. Incubate at room temperature for 5 minutes

3. Vigorously agitate the flask for 10 seconds Garring blows) 4. Add 10mI of DMEM+10% FBS to the flask

5. Tranfer 1/3 of the cell suspension into a fresh T75 flask containing 20ml of DMEM+10% FBS

6. Place the flask in an incubator at 37 degrees C/10%CO2 7. Feed as required, indicated by yellowing of the phenol red indicator in the medium, by aspirating and replacing the spent medium

8. Upon reaching confluence, passage the cells, using steps 1. to 6.

PREPARATION OF 577MF CONDITIONED MEDIUM CONTAINING WNT2B

1. At one day post passage, aspirate the medium from 577MF cells and replace with 30ml of fresh medium of the required formulation (e.g. HES medium, HES medium minus FGF, DMEM+10% FBS)

2. Place the flask in an incubator at 37 degrees C/10% CO2 3. At three days post passage, harvest the conditioned medium

4. Centrifuge the conditioned medium at 1000g for 5 minutes to pellet detached cells and debris

5. Collect the uppermost 2/3 of the medium and use immediately

DETECTION OF WNT2B SECRETION INTO CULTURE MEDtUWI BY 577MF CELLS

6. At one day post passage, aspirate the medium from 577MF cells and replace with 30ml of fresh Optimem (Gibco)

7. Place the flask in an incubator at 37 degrees C/10% CO2 8. At three days post passage, harvest the conditioned medium

9. Centrifuge the conditioned medium at 1000g for 5 minutes to pellet detached cells and debris

10. Transfer the uppermost 2/3 of medium to a VivaSpin 20 column (Sartorius stedim biotech) 11. Spin at 200Og for 20 minutes to concentrate the medium approximately tenfold

12. Run aliquots of the concentrated medium on a Western blot alongside size stadards

13. Probe the Western blot with i) anti-Wnt2b antibody (rabbit polyclonal, cat#38- 3500, Zymed Laboratories) and ii) an HRP conjugated anti rabbit secondary antibody

14. Visualise protein labelling by chemi-luminescence GENETIC MODIFICATION OF CELLS TO EXPRESS WNT2B FOR PRODUCTION OF CONDITIONED MEDIUM CONTAINING WNT2B

ENGINEERED CONSTITUTIVE EXPRESSION OF WNT2B

The coding sequence of Wnt2b [figure 3b or 4b] is inserted into the multiple cloning site of a plasmid vector containing a constitutive mammalian (e.g. PGK; figure 37, Ubiquitin C; figure 38, EF-1alpha; figure 39), viral (e.g. CMV; figure 40) or composite (e.g. CAG*; figure 41) promoter located 5' of it, a polyadenylation signal located 3' of it and containing a drug resistance gene for selection of transfected cells. Expression of the drug resistance gene may be driven from the same promoter as the inserted Wnt2b coding sequence by generating a bicistronic mRNA containing an internal ribosomal entry site (IRES; figure 42), or may be driven from a separate promoter to generate a second transcript.

The expression plasmid containing the Wnt2b coding sequence is transfected into a cell line or primary cells selected from the list below and the cells placed under drug selection added to the cells' normal growth medium. Clonal or non-clonal populations of drug resistant cells are expanded and then used to condition medium with Wnt2b protein for RPE production from human embryonic stem cells.

*pCAG is a composite promoter that combines the human cytomegalovirus immediate- early enhancer and a modified chicken beta-actin promoter and first intron (Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991 Dec 15;108(2):193-9).

ENGINEERED INDUCIBLE EXPRESSION OF WNT2B

The coding sequence of Wnt2b is inserted into an expression system that can be modulated by drug dosage, such as pTet-On-Advanced; figure 35 or pTet-Off-Advanced (cat# 630930 and 630934, respectively, Clontech/Takara Bio USA):

Tetracycline-controlled transactivators are expressed by regulator vectors: pTet-On- Advanced or pTet-Off-Advanced. Each transactivator is a fusion protein consisting of a re-engineered version of the bacterial Tet Repressor (TetR) and 3 repeats of a minimal VP 16 transcription activation domain. A response vector, pTRE-Tight; figure 43, containing an improved tetracycline response element (TRE) within the promoter that controls expression of the gene of interest. The regulator and response plasmids sequentially deliver each part of a Tet-Advanced System into the host cell line where they integrate into the genome to create a double- stable cell line. Once the double-stable line is established, the inducer doxycycline (Dox, a tetracycline derivative) controls the system in a dose-dependent manner, allowing expression levels of the target gene to be modulated.

A cell line or primary cell type is selected from the list below is transfected with either the pTet-On-Advanced or the pTet-Off-Advanced vector [Figure 44], for doxycylcine dependent induction or inhibition of expression, respectively. A stable, clonal cell line having incorporated the regulatory vector is then obtained using drug selection.

The Wnt2b coding sequence is ligated into the multiple cloning site of the response vector, pTRE-Tight. The response vector is then transfected into the stable clonal cell line containing the regulatory vector. Further drug selection is used to generate a double-stable transfected cell line that produces Wnt2b either in the presence of or in the absence of doxycycline, depending upon the choice of regulatory vector. The double-stable transfected cell line is expanded and then used to condition medium with Wnt2b protein for RPE production from human embryonic stem cells.

Claims

1 A method to enhance the proliferation and/or differentiation of progenitor retinal pigmented epithelial cells comprising: i) providing a cell culture preparation comprising: progenitor retinal pigmented epithelial cells, a cell culture surface and a cell culture medium wherein said medium is supplemented with a polypeptide that has the activity associated with a Wnt polypeptide; and ii) providing cell culture conditions that enhance the proliferation and/or differentiation of progenitor retinal pigmented epithelial cells.

2 A method according to claim 1 wherein said Wnt polypeptide is selected from the group consisting of: WNT 1, WNT 2, WNT 2b variant 1, WNT 2b variant 2, WNT 3, WNT 3A, WNT 4, WNT 5A, WNT 5B-1 , WNT 5B-2, WNT 6, WNT 7A, WNT 7B, WNT 8A₁ WNT 8B, WNT 9A, WNT 9B, WNT 1OA, WNT 1OB WNT 11, WNT 16 or WNT 16 variant 2.

3. A method according to claim 2 wherein said polypeptide is WNT 2b variant 1 or WNT 2b variant 2.

4. A method according to claim 2 or 3 wherein said polypeptide is selected from the group represented by the amino acid sequences in Figures 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a, 17a, 18a, 19a, 20a, 21a or 22a, or polypeptide variants thereof.

5. A method according to any of claims 1-4 wherein said Wnt polypeptide is exogenously added to said culture medium.

6. A method according to any of claims 1-4 wherein said cell culture medium comprises a cell line that produces said Wnt polypeptide.

7. A method according to claim 6 wherein said cell line naturally produces said Wnt polypeptide.

8. A method according to claim 6 wherein said cell line is transfected with a nucleic acid molecule that encodes said Wnt polypeptide.

9. A method according to claim 8 wherein said transfected nucleic acid molecule is operably linked to a promoter that regulates the expression of said nucleic acid molecule.

10. A method according to any of claims 1-9 wherein said cell culture medium further comprises at least one Wnt polypeptide antagonist to inhibit Wnt signalling thereby either directly or indirectly inducing differentiation of progenitor retinal pigmented epithelial cells.

11. A method according to claim 10 wherein said Wnt polypeptide antagonist is an antibody.

12. A method according to claim 10 wherein said Wnt polypeptide antagonist is a natural inhibitor of Wnt signalling.

13. A method according to claim 12 wherein said natural Wnt polypeptide inhibitor is selected from the group consisting of: CER, WIF 1 , SFRP1, SFRP2, SFRP4, SFRP5, FRZB, DKK1, DKK2, DKK3 (transcript variants 1-3), DKK4 or DKKL1..

14. A method according to claim 13 wherein said Wnt polypeptide inhibitor is represented by the amino acid sequence in Figure 23a, 24a, 25a, 26a, 27a, 28a, 29a, 30a, 31a, 32a, 33a, 34a, 35a or 36a or functional variant thereof.

15. A cell culture comprising: progenitor retinal pigmented epithelial cells and a cell culture medium wherein said medium is supplemented with a polypeptide that has the activity associated with a Wnt polypeptide.

16. A cell culture according to claim 15 wherein said cell culture medium comprises a polypeptide selected from the group consisting of: WNT 1 , WNT 2, WNT 2b variant 1 , WNT 2b variant 2, WNT 3, WNT 3A₁ WNT 4, WNT 5A, WNT 5B-1 , WNT 5B-2, WNT 6, WNT 7A, WNT 7B, WNT 8A, WNT 8B, WNT 9A, WNT 9B, WNT 10A, WNT 10B WNT 11 , WNT 16 or WNT 16 variant 2.

17. A cell culture according to claim 16 wherein said polypeptide is WNT 2b variant 1 or WNT 2b variant 2.

18 A cell culture according to claim 16 or 17 wherein said polypeptide is selected from the group represented by the amino acid sequences in Figures 1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 12a, 13a, 14a, 15a, 16a, 17a, 18a, 19a, 20a or 21a, or polypeptide variants thereof.

19. A cell cuture according to any of claims 15-18 wherein said cell culture medium comprises at least one Wnt polypeptide antagonist to inhibit Wnt signalling thereby, either directly or indirectly, inducing differentiation of progenitor retinal pigmented epithelial cells.

20. A cell culture according to claim 19 wherein said Wnt polypeptide antagonist is an antibody.

21. A cell culture according to claim 19 wherein said Wnt polypeptide antagonist is a natural inhibitor of Wnt signalling.

22. A cell culture according to claim 21 wherein said Wnt polypeptide inhibitor is selected from the group consisting of: CER, WlF 1 , SFRP1 , SFRP2, SFRP4, SFRP5, FRZB, DKK1, DKK2, DKK3 (transcript variants 1-3), DKK4 or DKKL1.

23. A cell culture according to claim 22 wherein said Wnt polypeptide inhibitor is represented by the amino acid sequence in Figure 23a, 24a, 25a, 26a, 27a, 28a,29a,

30a, 31a, 32a, 33a, 34a, 35a or 36a, or functional variant thereof.

24. The use of Wingless [Wnt] polypeptide in the stimulation of proliferation and/or differentiation of progenitor retinal pigmented epithelial cells [PRPE].

25. A cell line transfected with a nucleic acid molecule selected from the group consisting of. i) a nucleic acid molecule comprising or consisting of a nucleic acid sequence as represented by the sequences presented in Figures 1b, 2b, 3b, 4b, 5b, 6b, 7b, 8a, 9b, 10b, 11b, 12b, 13b, 14b, 15b, 16b, 17b, 18b,

19b, 20b, 21b or 22b; ii) a nucleic acid molecule comprising a sequence that hybridizes under stringent hybridization conditions to the molecules in i) above and encodes a polypeptide with the activity associated with a Wnt polypeptide, wherein said nucleic acid molecule is operably linked to a control sequence that regulates the expression of said nucleic acid molecule.

26. A cell line according to claim 25 wherein said cell line is transfected with a nucleic acid molecule comprising of a nucleic acid sequence as represented in Figure 3b or 4b.

27. The use of a cell according to the invention in the stimulation of proliferation and/or differentiation of progenitor retinal pigmented epithelial cells [PRPE].

28. Use according to claim 27 wherein said cell is transfected with a nucleic acid molecule that encodes a polypeptide with the activity associated with Wnt 2b.

29. Use according to claim 28 wherein said nucleic acid molecule comprises or consists of the nucleic acid sequence represented in Figures 3b or 4b.

30. A method to treat a subject suffering from macular degeneration comprising surgically administering a differentiated retinal pigmented epithelial cell prepared by the method according to any of claims 10-14.