Nothing Special   »   [go: up one dir, main page]

WO2015113110A1 - A method for the purification of eye lens cells - Google Patents

A method for the purification of eye lens cells Download PDF

Info

Publication number
WO2015113110A1
WO2015113110A1 PCT/AU2015/000046 AU2015000046W WO2015113110A1 WO 2015113110 A1 WO2015113110 A1 WO 2015113110A1 AU 2015000046 W AU2015000046 W AU 2015000046W WO 2015113110 A1 WO2015113110 A1 WO 2015113110A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
lens
cell
eye lens
purified
Prior art date
Application number
PCT/AU2015/000046
Other languages
French (fr)
Inventor
Michael O'connor
Patricia Murphy
Original Assignee
University Of Western Sydney
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2014900269A external-priority patent/AU2014900269A0/en
Application filed by University Of Western Sydney filed Critical University Of Western Sydney
Publication of WO2015113110A1 publication Critical patent/WO2015113110A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0618Cells of the nervous system
    • C12N5/0621Eye cells, e.g. cornea, iris pigmented cells

Definitions

  • the present invention relates to a method for purifying a population of cells, and to preparations of the cells and uses of them.
  • Presbyopia Hardening of the ocular lens (presbyopia) affects people from approximately age 45. This results in reduced ability to change focus between near and far objects (i.e., reduced accommodation), and affects many aspects of daily life including reading and driving. Between 500 million and 1 billion people worldwide suffer from presbyopia. Current treatments typically consist of corrective spectacles or contact lenses, particularly for close vision.
  • multi-focal contact lenses have been developed that provide some vision correction for people with presbyopia.
  • these contact lenses are not available to all patients due to incompatible prescription type or too large pupil size.
  • treated patients often discontinue contact lens use due to inherent glare and halos that affect daily activities, as well as discomfort, dryness, infection, ongoing costs and daily handling requirements.
  • Cataract-induced blindness affects 100 million people globally and currently can only be treated surgically.
  • Cataract surgery involves removal of the non-transparent biological lens and implantation of an intraocular lens (IOL).
  • IOL intraocular lens
  • cataract surgery leads to loss of accommodation, necessitating the use of approaches to try and redress this complication such as multifocal spectacles or IOLs.
  • IOL intraocular lens
  • cataract operations are performed annually costing in excess of $326 million. Patient numbers and costs are each 10 to 20 times higher in the United States, Europe and Asia.
  • cataracts Whilst advancing age is a key risk factor for cataract, other cataracts that occur in children or youths (such as congenital or traumatic paediatric cataract) can result in significant life-long learning, behavioural and psychological issues for the sufferer. This is particularly true in developing countries where paediatric lens removal can occur without optical correction (aphakia) due to difficulties surgeons face choosing IOLs for infant eyes that will grow significantly in the first years of life (i.e., growth that is not easily matched by fixed size, non-accommodating IOLs).
  • PCO posterior capsule opacification
  • hPSCs human pluripotent stem cells
  • Lentoids comprise of a mixture of lens fibre cells (LFCs) and lens epithelial cells (LECs) and do not correctly mirror the complex architecture of the lens, namely, a monolayer of anterior epithelial cells overlying a compacted mass of parallel-aligned, elongated lens fibre cells.
  • Non-lens cells are also produced via this method, and any remaining undifferentiated hPSCs have the potential to generate teratomas and may interfere with the outcome of in vitro differentiation protocols.
  • Fluorescence activated cell sorting can be employed to deplete
  • the present invention relates to the identification of cell surface markers that are expressed on the outer cell membrane of particular eye lens cells whilst either being not expressed or simultaneously only relatively lowly expressed on other unwanted tissues or cell types by comparison, and embodiments as described herein can provide for the relatively rapid purification of large numbers of the lens cells.
  • a method for the purification of eye lens cells and/or progenitor cells thereof from a mixed population of cells comprising isolating cells on the basis of outer cell membrane expression of at least one marker selected from the group consisting of ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 from the mixed cell population, and collecting the isolated cells.
  • the mixed population of cells can, for example, comprise a mixed population of embryonic cells, postnatal, juvenile or adult cells.
  • the method may further comprise providing the mixed population of cells by inducing pluripotent stem cells to differentiate into the lens cells and/or progenitor cells thereof.
  • the selected marker is RORl .
  • the selected marker is GPR161.
  • the selected marker is CD81.
  • the selected marker is ODZ3.
  • the selected marker is SLC7A11.
  • the selected marker is SLC16A1.
  • the selected marker is SLC23A2.
  • the marker is selected from RORl, and GPR161.
  • a method for the purification of eye lens cells and/or progenitor cells thereof from a mixed population of cells comprising isolating cells on the basis of outer cell membrane expression of RORl and/or GPR161 from the mixed cell population, and collecting the isolated cells.
  • the eye lens cells and/or progenitor cells thereof express the selected marker and one or more other of the markers from the group consisting of RORl, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2.
  • the eye lens cells or progenitor cells thereof express a majority of the markers and most typically, all of the markers.
  • the purified eye lens cells are LECs and/or progenitor forms of LECs.
  • the purified eye lens cells can comprise LECs and/or LFCs, and/or progenitor forms of LECs and/or LFCs.
  • the purified cells comprise LFCs or progenitor forms of LFCs, the LFCs or progenitor cells thereof may comprise or consist of immature LFCs and/or cells that are in the process of
  • the cells purified in accordance with a method embodied by the invention will consist of purified populations of LECs, progenitor forms of LECs and/or early differentiating LECs.
  • LECs and/or progenitor forms of LECs purified in accordance with a method embodied by the invention express at least RORl from the group of cell surface markers.
  • LFCs and/or progenitor forms of LECs purified in accordance with the invention express at least GPR161 from the group of the cell surface markers.
  • the isolation of the cells comprises contacting cells in the mixed cell population with a respective binding agent for binding to the selected marker and separating the cells to which the binding agent has bound from the mixed population.
  • the binding agent can be any suitable ligand for the selected marker, or for instance, an antibody or a binding fragment thereof.
  • the eye lens cells and/or progenitor cells thereof can be purified from the mixed population of cells utilising a binding agent for a single one of the cell surface markers (e.g., RORl) in at least some embodiments of the invention a plurality of binding agents may be utilised, each of the binding agents binding to a different one of the cell surface markers, respectively.
  • one binding agent which binds to RORl and another binding agent which binds to GPR161 (and so on) can be employed.
  • the cells bound by the respective binding agent are separated from the mixed population of cells by flow cytometry or magnetic separation.
  • Any suitable magnetic separation protocol may be employed but typically, the magnetic separation comprises magnetic activated cell sorting (MACS).
  • MCS magnetic activated cell sorting
  • a purified population of eye lens cells and/or progenitor cells thereof having outer cell membrane expression of at least one marker selected from the group consisting of RORl, GPR161, CD81, ODZ3, SLC7A11, SCL16A1 and SLC23A2.
  • progeny cells from cell preparations embodied by the invention.
  • an assay for screening the effect of a test agent on eye lens cells comprising:
  • the assay is a toxicology assay. In another embodiment the assay is a drug screening assay for assessing the effect of a therapeutic or putative drug on the cells.
  • the cells purified or utilised in accordance with a method embodied by the invention can be normal cells, aberrant cells (e.g., diseased cells), or mixtures of the foregoing.
  • a method for providing a lentoid comprising culturing purified cells embodied by the invention under conditions suitable for the generation of the lentoid and for a period of time to generate the lentoid.
  • the method can also comprise removing the lentoid from the cultured cells for further culturing and/or study.
  • embodiments of a purification method as described herein may provide for the preparation of large numbers of LECs or LFCs (particularly embryonic LECs or LFCs) in high purity (e.g., pure or substantially pure cell preparations) which can be subsequently utilised in drug screening and toxicology assays, or for being cultured for research or other purposes.
  • at least some forms of the invention provide for the purification of the cells rapidly, inexpensively and without the need for access to complex equipment such as flow cytometers.
  • methods for purification of the cells may be carried out using common, readily available laboratory equipment.
  • Figure 1 A) GenePaint analysis of gene lists (generated using the Excel-based macro) identified ROR1 (shown), GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 (not shown) as having highly-restricted lens expression patterns in mouse embryos approximately E14.5.
  • Figure 2 A) A light micrograph showing MACS-purified lens cells in culture (derived from differentiating CA1 human embryonic stem cell cultures; lOx
  • Figure 3 Graph showing survival of hLECs purified in accordance with an embodiment of the invention exposed to pure water in a cell death assay.
  • Figure 4 Correlation analysis showing the gene expression profiles of purified human lens cell samples are more similar to early embryonic mouse stages than later embryonic stages or postnatal stages.
  • Figure 5 Expression of selected key lens genes by purified human lens cells.
  • A, B histograms of gene expression for Cufflinks FPKM and HT-seq counts.
  • Figure 6 Expression of key lens genes by purified human lens cells.
  • A,B histogram of gene expression for Cufflinks FPKM and HT-seq counts, respectively.
  • Figure 7 A-G) El 4.5 mouse in situ hybridisation data showing lens specificity of 7 cell membrane proteins for human lens cell purification that are expressed by adult primary human lens epithelial cells. H) RNA-Seq expression levels show all of lens cell purification markers ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 are expressed in ROR1 -purified human lens epithelial cells..
  • the mixed population of cells utilised in a purification method embodied by the invention is preferably a population of differentiated (or differentiating) stem cells (e.g., pluripotent stem cells)
  • the cells may be obtained from various other sources including adult cells, cultured or non-cultured primary lens cells, lens cell lines, and for instance, transdifferentiation of iris or corneal cells (e.g., newts, frogs and other sources).
  • Primary lens cells for example, can be obtained directly from isolated eye lens tissue and subjected to a method as described herein.
  • Pluripotent stem cells which may be induced to differentiate into lens cells or progenitor forms thereof purified in accordance with the invention include WA01 (HI) human embryonic stem cells
  • ROR1 Receptor Tyrosine Kinase-like Orphan Receptor 1
  • human and mouse nucleic acid sequences are available on publicly accessible databases (e.g., Human: Uniprot ID. Q001083592, NM_001083592 (mRNA), NP 001077061 (AA); Mouse: Uniprot ID. Q9Z139, NM_013845 (mRNA), NP_038873 (AA)).
  • GPR161 is a previously reported G-protein coupled receptor (e.g., Human: Uniprot ID. Q8N6U8; NM_001267609 (mRNA), NP_00125438 (AA); Mouse: Uniprot ID. B2RPY5,
  • NM_001081126 mRNA
  • NP_0010745959 AA
  • CD81 has been reported to complex with integrins and to appear to promote muscle cell fusion and support myotube maintenance, and may be involved in signal transduction (e.g., Human:
  • ODZ3 belongs to the tenascin family and has been reported to possibly be a cell signal transducer (e.g., Human: Uniprot ID. Q9UKZ4, NM_001163278 (mRNA), NP_001156750 (AA);
  • a cell signal transducer e.g., Human: Uniprot ID. Q9UKZ4, NM_001163278 (mRNA), NP_001156750 (AA);
  • SLC16A1 has been reported to be a proton-linked monocarboxylate transporter for transport of monocarboxylates such as lactate and pyruvate across the plasma membrane (e.g., Human: Uniprot ID. P53985, NM_ 001166496 (mRNA), NP 001159968 (AA); Mouse: Uniprot ID. P53986, NM_ 009196 (mRNA), NP_033222 (AA)).
  • SLC23A2 has been reported to be a sodium-dependent ascorbic acid (vitamin C) transporter (e.g., Human: Uniprot ID.
  • Cells that may be purified in accordance with a method of the invention include LECs, progenitors of LECs and early-differentiating LFCs (e.g., expressing one or more of ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2).
  • eye-differentiating encompasses immature LFCs
  • progenitor encompasses precursor cells that give rise to LECs and/or LFCs.
  • lens progenitor cells within the preplacodal region of late gastrulation stage embryos.
  • the cells When embryonic cells are purified in accordance with the invention the cells will typically be LECs, early-differentiating LFCs or progenitor cells thereof.
  • the embryonic cells will be at least equivalent to lens progenitor cells in the preplacodal region of gastrula stage embryos, or later stage lens progenitor and lens cells.
  • Various purification techniques may be utilised to purify lens cells expressing the selected cell surface marker(s) in accordance with the invention including, but not limited to, FACS and magnetic separation techniques.
  • FACS Fluorescence Activated Cell Sorting
  • the mixed cell population may be incubated with a respective primary antibody specific for the selected marker prior to washing and incubating the cells with a secondary antibody specific for binding to the primary antibody and which is labelled with a fluorescent marker (e.g., phycoerythrin or FITC) for separation of the labelled cells utilising a flow cytometer employing known protocols.
  • a fluorescent marker e.g., phycoerythrin or FITC
  • Magnetic separation techniques that may be employed in a method of the invention can utilise paramagnetic substrate particles coated with a respective antibody or other binding agent for binding either directly or indirectly to the selected cell surface marker(s) expressed on the target cell(s).
  • Indirect binding may, for example, be achieved by the binding of a secondary antibody on the magnetic particle to a primary antibody bound to the selected marker(s) expressed on the outer membrane of the target cell.
  • the magnetic particle may be coupled to the target cell via streptavidin provided on the particle linking to a biotinylated primary antibody or other binding agent bound to the selected marker(s) expressed on the cell, and all such variations are expressly encompassed.
  • the cells are placed in a magnetic field provided by fixed magnet(s) to which the magnetic particles are attracted allowing those cells not bound by the magnetic particles to be e.g., eluted, decanted, or otherwise physically separated from the magnetically bound cells.
  • Suitable such solid substrates include plastics (e.g., polypropylene and other suitable plastics) and synthetic resins, and beads of e.g., latex, polystyrene, dextran, agarose, sepharose, glass and synthetic resins as described above.
  • An antibody, binding fragment or other binding agent as described herein can be bound to a solid substrate covalently utilizing commonly employed amide or ester linkers or, for instance, by adsorption. Protocols for the preparation of such solid substrates for affinity separation techniques are for instance described in Current Protocols in Molecular Biology - Ausubel FM. et al, Wiley-Interscience, 1988 and subsequent updates thereof.
  • collected target cells may be washed one or more times with a suitable physiologically acceptable buffer and subjected to additional round(s) of separation in order to further increase the purity of the isolated target lens cells.
  • a combination of cell purification techniques as described herein may be utilised (e.g., one or more rounds of magnetic separation followed by FACS).
  • Magnetic separation e.g., MACS
  • Magnetic separation techniques that may be employed include DynabeadTM and MACS techniques.
  • MACS purification has the advantage over DynabeadTM cell purification in that commercially available magnetic nanoparticles used in MACS are biodegradable, and so there is no need for use of a releasing agent to separate the magnetic beads from the captured lens cells.
  • the binding agent used in a method of the invention will be an antibody.
  • the antibody can be polyclonal or monoclonal although the latter is preferred.
  • the production of polyclonal antibodies and monoclonal antibodies is well established in the art (e.g., see Antibodies, A Laboratory Manual. Harlow & Lane Eds. Cold Spring Harbour Press, 1988).
  • a mammal such as a sheep, goat or rat is immunized with an antigenic fragment of the target protein (e.g., human ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 or SLC23A2) and anti- sera is subsequently isolated from the mammal prior to purification of the antibodies generated against the antigen by standard affinity chromatography techniques such as Sepharose-Protein A chromatography.
  • the mammal is periodically challenged with the relevant antigen to establish and/or maintain high antibody titer.
  • B lymphocytes can be isolated from the immunized mammal and fused with immortalizing cells (e.g., myeloma cells) using somatic cell fusion techniques (e.g., employing polyethylene glycol) to produce hybridoma cells (e.g., see Handbook of Experimental Immunology, Weir et al Eds. Blackwell Scientific Publications. 4th Ed. 1986).
  • immortalizing cells e.g., myeloma cells
  • somatic cell fusion techniques e.g., employing polyethylene glycol
  • hybridoma cells e.g., see Handbook of Experimental Immunology, Weir et al Eds. Blackwell Scientific Publications. 4th Ed. 1986.
  • the selection of hybrid cells may be achieved by culturing cells in hypoxanthine-aminopterin-thymidine (HAT) medium, and selected hybridoma cells then screened for production of antibodies specific for the target protein by enzyme linked immunosorbant assay (ELISA) or other immunoassay
  • Antibodies that may be utilised for binding to ROR1 for the purification of lens cells or progenitor cells thereof in accordance with the invention include AF2000 (RandD Systems, Inc., Minneapolis, MN 55414, USA). Antibodies for binding to GP161 may for instance be selected from ab58679 (Abeam, Cambridge, MA 02139- 1517, USA), ⁇ 1734983 (Antibodies Online Inc., Atlanta, GA 30338, USA), orb 157327 (Biorbyt LLC, San Francisco, CA 94104, USA), MC-331 (MBL
  • Antibodies for binding to CD81 may for instance be selected from AP6631 (Abgent, Inc., San Diego, CA 92124, USA), orb36798 (Biorbyt LLC, San Francisco, CA 94104, USA), OAAB03662 (Aviva Systems Biology Corp., San Diego, CA 92121, USA), GTX81801 (GeneTex Inc., Irvine, CA 92606, USA), and PA5-1358 (Thermo Fisher Scientific Inc., Rockford, IL 61101, USA).
  • Antibodies for binding to ODZ3 include orbl58049 (Biorbyt LLC, San Francisco, CA 94104, USA), TA321274 (CliniSciences, Nanterre 92000, FR), and sc-136920 (Santa Cruz, Inc., Dallas, TX 75220, USA).
  • Antibodies for binding to SLC7A11 may for instance be selected from antibodies abl 11822, ab60171, ab99059, abl 12403, and ab84171 (Abeam, Cambridge, MA 02139-1517, USA),
  • Antibodies for binding to SLC16A1 may for instance be selected from ab85021 and ab90582 (Abeam, Cambridge, MA 02139-1517, USA), LS- C335287 (LSBio, Inc., Seattle, WA 98121, USA), LS-C341521 (LSBio, Inc., Seattle, WA 98121, USA), and HPA003324-100UL (Sigma-Aldrich, LLC, St. Louis, MO 63103, USA).
  • Antibodies for binding to SLC23A2 include antibodies sc-9927, sc-30113, and sc-376090 (Santa Cruz, Inc., Dallas, TX 75220, USA).
  • binding agents which may be used in a method of the invention besides whole antibodies include binding fragments of antibodies and other proteinaceous agents that bind to the selected surface marker(s), and streptavidin, biotin and the like.
  • binding fragment expressly includes within its scope Fab and (Fab') 2 fragments obtainable by papain or pepsin proteolytic cleavage respectively, variable domains of antibodies (e.g., Fv fragments), and antibody single chain variable fragments (scFvs) and multimer forms thereof such as bivalent scFvs (e.g., bivalent and diabodies), trivalent scFvs (triabodies) and tetravalent scFvs (tetrabodies), that bind to the target protein.
  • Strategies for identifying other proteinaceous agents that may be used in a method for the purification of lens cells as described herein include large scale screening techniques. For instance, phage display library protocols provide an efficient way of testing a vast number of potential peptide
  • Phage display libraries express random transgenic peptides or antibody variable domain(s) of known length on the surface of the selected bacteriophage. Each phage clone displays a distinct such peptide sequence.
  • the peptide sequences are fused with major or minor coat proteins of the selected phage type and can be produced by inserting random oligonucleotides in DNA encoding the coat protein, transfecting the resulting construct into a suitable host bacterial strain, and generating phage particles upon superinfection of the bacterial strain with helper phage.
  • Peptides which bind to the selected cell surface marker(s) can, for instance, be identified by contacting lens cells expressing the target protein to identify phage clones in the library which bind to the protein.
  • Unbound phage is washed away and the remaining bound phage is recovered.
  • the pool of bound phage can be enriched by subjecting the bound phage to a number of such biopanning cycles, wherein the bound phage is collected and amplified utilising suitable host bacteria before being subjected to the next cycle.
  • the sequence of the binding peptide of an isolated phage clone may then be identified by sequencing the relevant coat protein of the clone, and comparing that sequence with the known sequence for the native phage coat protein.
  • Drug screening and toxicology assays in accordance with the invention will typically involve culturing cells purified as described herein in the presence of a known or putative drug or toxicant for a period of time sufficient for the effects of the drug or toxicant to be observed directly (e.g., visually or with the aid of a microscope) or otherwise be detectable such as by staining of the cells or subjecting the cells or cell culture supernatant to appropriate analysis.
  • the cells can be cultured with the drug or toxicant alone or in combination with one or more other agents (e.g., cytokines, cell regulatory agents, cell differentiation agents, growth factors etc.) and/or cell types.
  • the cells may be cultured in such assays for e.g., minutes, hours, overnight or days, and may require the culture medium to be changed, refreshed or supplemented during the culture period.
  • the cells utilised in toxicity screening or drug testing assays as described herein can be normal cells or aberrant cells purified in accordance with a method embodied by the invention.
  • aberrant cells can, for example, be cells that are deficient in one or more characteristics compared to normal cells or be diseased cells (e.g., cancer cells, abnormally growing LECs, cells which causing primary or secondary cataract, cells affected by age-related cataract, and cells with congenital or acquired mutation(s) that cause cataract, etc).
  • an assay as described herein may be utilised for screening for an anti-posterior capsule opacification (PCO) drug.
  • PCO arises from residual primary human LECs that are not removed during primary cataract surgery. These residual lens epithelial cells migrate along the interior surface of the lens capsular bag (that holds the intraocular lens implanted during cataract surgery). Once the residual primary LECs reach the posterior of the capsular bag the local growth factor environment causes the cells to elongate and wrinkle the capsule which causes light scatter (i.e., PCO). Once PCO occurs vision can only currently be restored by laser- based cutting of the posterior lens capsule (which then falls away from the capsular bag but remains in the eye).
  • an assay as described herein may be utilised to identify one or more compounds that kill, inhibit the migration of, render mitotically inactive, or differentiate to LFCs residual LECs left after cataract surgery..
  • the cells may be cultured in the wells of a tissue culture plate (e.g. a 12, 24 or 96 well or other plate or dish) and exposed to the test agent for a predetermined period of time prior to assessing the effect of the test agent on the cells.
  • a tissue culture plate e.g. a 12, 24 or 96 well or other plate or dish
  • the cells will be cultured at a density in a range of from tens of cells per well (e.g., for 96, 384, 1536 well plates) up to hundreds of thousands of cells per well or dish (e.g., for multi-well plates).
  • Suitable culture medium for culturing the cells may be selected from typical base media (e.g., RPMI, DMEM, DMEM, F12, F12, Ml 99, etc) with additional growth supplements (e.g., fetal bovine serum, bFGF, ocular fluids such as aqueous fluid or vitreous fluid, etc) with or without antibiotics and/or anti-fungal agents.
  • typical base media e.g., RPMI, DMEM, DMEM, F12, F12, Ml 99, etc
  • additional growth supplements e.g., fetal bovine serum, bFGF, ocular fluids such as aqueous fluid or vitreous fluid, etc
  • antibiotics and/or anti-fungal agents e.g., antibiotics and/or anti-fungal agents.
  • the cells may be cultured in the presence of the test agent for any predetermined appropriate time in order for the effect(s) of the test agent(s) to be observed such as from 1 minute to 1 or more hours (e.g., 2, 3, 4, 5, 6, 7 or 8 hours, overnight, 1 day, 2 days etc.) depending on the nature and concentration of the test agent.
  • fresh culture media can be added to the test wells or media containing the test agent can be aspirated from the wells and replaced with fresh media with or without the test agent.
  • the effect of the test agent on the cells can be evaluated by any manner that quantifies the loss of live cells and/or the appearance of dead cells such as by MTT growth assay, detection of live and dead cell numbers such as by involving propidium iodide staining of viable cells or use of other cell viability dyes or stains (e.g., nigrosin, LIVE/DEAD® Cell Viability Assay reagents, etc), visual evaluation of cell
  • Test agents that may be assessed by a method as described herein utilising purified cells embodied by the invention include putative anti- primary or secondary cataract drugs as well as inhibitors or agonists of cell signalling pathway (e.g., kinases, receptors etc.), ion channels, enzymes, and proteasome and proteasome pathway members, etc.
  • cell signalling pathway e.g., kinases, receptors etc.
  • ion channels e.g., kinases, receptors etc.
  • enzymes e.g., enzymes, and proteasome and proteasome pathway members, etc.
  • Purified cells embodied by the invention may also be cultured under suitable conditions to generate lentoid(s). This may involve culturing the cells at cell densities sufficient for the formation of a lentoid, and (for example) supplementing culture media with one or more cell differentiating agents (e.g., FGF family members such as bFGF, and/or other growth factors, ocular fluids such as vitreous fluid, etc) for promoting the formation of the lentoid.
  • cell differentiating agents e.g., FGF family members such as bFGF, and/or other growth factors, ocular fluids such as vitreous fluid, etc
  • lentoid in the context of the present invention is to be taken to encompass a 3 -dimensional lens like structure of tissue(s) comprising populations of cells selected from LECs, LFCs, early differentiating LFCs, and progenitor cell forms of the aforementioned cells.
  • a lentoid may be prepared by culturing purified cells embodied by the invention at a cell concentration and in the presence of bFGF sufficient for promoting the development of the lentoid as described further below.
  • the cells will be cultured until confluent.
  • the cell culture medium will generally contain bFGF at a concentration of at least 1 ng/ml, more usually at least 20 ng/ml and most preferably in a range of from 50 ng/ml to about 100 ng/mL.
  • the culture medium utilised may, for example be selected from RPMI, DMEM, DMEM:F12, F12, Ml 99, etc.
  • the lens cells may be purified in accordance with the invention to high levels of purity for use in drug screening and toxicology assays or for other uses such as described herein.
  • the term "purified” in the context of the invention is to be taken to encompass populations of the target lens cell(s) that are of a higher purity than the mixed cell population from which they were isolated.
  • Purified preparations of the lens cells expressing the selected cell surface marker(s) include preparations that have a purity level of at least about e.g., 80%, 85%, 90% or 95% or greater (e.g., 96%, 97%, 98%, 99% or 100%).
  • non-RORl i.e., ROR1 "
  • non-GPR161 i.e., GPR161 "
  • non-CD81 i.e., CD81 "
  • non-ODZ3 i.e., ODZ3 "
  • non-SLC7Al 1 i.e., SLC7A11 "
  • non-SLC16Al i.e., SLC16A1 "
  • non-SLC23A2 i.e., SLC23A2 " ) expressing cells.
  • Such contaminating cells may comprise e.g., ⁇ 10% of the cell preparation (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% or less) depending on the conditions the cells experience.
  • the cells purified in accordance with a method embodied by the present invention may be obtained from various animals including but not limited to birds, amphibians (e.g., frogs and newts), and mammals such as rabbits and members of the rodent (e.g., mice, rats and hamsters), bovine, ovine, equine, porcine, canine, feline and primate (e.g., chimpanzees, Rhesus monkeys and baboons) families, and humans.
  • amphibians e.g., frogs and newts
  • mammals such as rabbits and members of the rodent (e.g., mice, rats and hamsters), bovine, ovine, equine, porcine, canine, feline and primate (e.g., chimpanzees, Rhesus monkeys and baboons) families, and humans.
  • the cells will be human cells (e.g., primary human eye lens cells, embryonic cells, etc).
  • the cells are cells that are obtained by inducing human pluripotent stems cells to differentiate into lens cells, and may include or comprise differentiated lens cells and/or precursor forms thereof in the lens cell lineage that express the selected cell surface marker(s) i.e., RORl, GPR161, CD81, 0DZ3, SLC7A11, SLC16A1 and/or SLC23A2).
  • the 3 stage growth factor protocol of Yang et al, 2010 was assessed for its ability to produce crystallin-expressing human eye lens epithelial cells (LECs) and eye lens fibre cells (LFCs) in culture using CA1 human pluripotent stem cells (hPSCs)
  • LECs human eye lens epithelial cells
  • LFCs eye lens fibre cells
  • hPSCs CA1 human pluripotent stem cells
  • hPSCs human pluripotent stem cells
  • Human pluripotent cell medium e.g., mTeSRl, StemCell Technologies
  • PBS Phosphate buffered saline
  • cell viability stain e.g. Nigrosin
  • an Excel- based macro was developed using visual basics that organizes gene expression data based on relative expression levels across multiple samples. The macro was written to perform a primary sort of the rows within 3 columns of the input worksheet in order to rank genes from highest to lowest expression across these 3 samples. Where analysis of greater than 3 samples is required, a secondary sort of the rows in an additional 3 columns can be performed in comparison to the first 3 columns. This enables ranking of additional replicate array data from the same or a different cell type or tissue.
  • user-downloaded data from public gene expression repositories e.g., the Gene Expression Omnibus, GEO
  • GEO Gene Expression Omnibus
  • the minimum information required for macro function being a unique gene identifier in alphabetical order (e.g., Affymetrix ID) and an associated expression value (e.g., present and absent calls; expression value; etc.) from each microarray sample of interest.
  • the GEO dataset GSE2256 (Hawse et al., 2005) was analysed via the macro to identify membrane proteins expressed by LECs. Output gene lists from the macro were then analysed via a pipeline of publically-available tools to identify new lens cell biomarkers, including: gene ontology assessment (via the DAVID webserver, National Institute of Alergy and Infectious Diseases (NIAD), National Cancer Institute,
  • LECs and LFCs e.g., 44 cell surface receptors identified in the LEC group, 15 in the LFC group, and 60 common to both tissues.
  • In situ hybridisation expression patterns in mouse embryos are available via the Genepaint webserver, Max Plank Institute for Biophysical Chemistry, Goettingen, Germany. In situ hybridisation allows all stages of lens development to be examined i.e., lens pit (E10.5), lens vesicle (El 1.5), primary lens fiber cells (E12.5), and differentiating secondary lens fiber cells (E14.5).
  • lens pit E10.5
  • lens vesicle El 1.5
  • primary lens fiber cells E12.5
  • differentiating secondary lens fiber cells E14.5.
  • the culture system used herein described further below best reflects a stage where immature epithelium and
  • stage E14.5 was assessed.
  • ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 genes were identified to have gene expression patterns highly restricted to the eye lens during embryonic development.
  • ROR1 showed a strong anterior lens epithelial gene expression with little expression in other tissues (see Fig. 1 and Fig. 6).
  • the immortalised foetal human lens epithelial cell line FHL124 was assessed for expression of RORl and GPR161 via polymerase chain reaction (PCR), and both transcripts were found to be expressed. Additional assessment of human lens cell differentiation samples at different time points also showed expression of both transcripts. RNA-seq analysis also showed all 7 lens cell purification markers RORl, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 to be expressed in purified human lens cells (Fig. 6), as further described below in Example 4.
  • PCR polymerase chain reaction
  • PBS Phosphate buffered saline
  • BSA BSA
  • Flow cytometry detection of RORl labelled cells determined the total percentage of cells expressing RORl in day 18 cultures prior to MACS to be -60%. Once cells were purified, flow cytometry showed one dominant population of RORl expressing cells with up to -99% of MACS purified cells RORl positive. This is further supported by flow cytometry assessment of positively collected RORl + cells showing 99% expression of the LEC marker aB-crystallin. PCR analysis of sorted cells showed a higher expression of aB-crystallin in RORl collected cells as opposed to the negative and unsorted fractions. Additionally, PB3-crystallin (a marker of lens fibre cell lineage) was more highly expressed in unsorted and RORl negative cell populations.
  • RORl can be used to positively select LECs from a mixed population of cells.
  • antibodies specific for other of the cell surface markers GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 may also, or alternatively, be used for the purification of eye lens cells in accordance with the invention due to their high and relatively restricted embryonic lens expression pattern, and their relatively high expression in purified human lens cells.
  • bFGF Basic fibroblast growth factor
  • the reported 3-stage lens cell differentiation method employs high concentrations of bFGF in the final stage of the protocol together with the addition of Wnt-3a. As that system produced contaminating fibre cells and lentoid structures, low concentrations of bFGF was employed for maintenance of hPSC-derived LECs and to avoid stimulation of differentiation into lens fiber cells.
  • Cells purified for RORl expression by MACS as described above using CA1 and/or MEL1 human embryonic stem cells were cultured at differing cell densities in 1 ml of RPMI media containing 20 ng/nL bFGF in Matrigel-coated wells of a 24 well plate with 10 ⁇ Ri on the day of plating. The medium was then changed the next day with RPMI containing 20 ng/mL bFGF without Ri, and changed with this medium approximately twice weekly as necessary.
  • Cells may be cultured in DMEM, DMEM:F12, F12, Medium 199 (M199) or similar medium instead of RPMI . The cultured cells were assessed for the appearance of lentoids and micrographs taken.
  • the MACS purified RORl + cells can be readily cultured at a concentration of 4xl0 5 cells/well of a 24 well plate for subsequent use as required (or approximately equivalent seeding densities of different sized culture dishes).
  • the MACS purified cells optimally require neighboring cells to proliferate and can be maintained in low bFGF conditions.
  • high densities of the purified cells can stimulate lentoid production. This may also mirror events in the native eye lens as throughout life the epithelium proliferates providing an ongoing supply of secondary lens fibre cells which constantly encircle the lens.
  • these results also show that higher concentrations of bFGF may be used to stimulate LEC differentiation to fibre cells in a controlled setting.
  • the purified hLECs were exposed to pure water in a cell death assay. Water was chosen as it has previously been used in a clinical trial to remove primary human lens epithelial cells during cataract surgery, although with no long-term reduction in the rate of PCO (Rabsiler et al. 2007. Br J Ophthalmology 91 :912-5).
  • purified hLECs were seeded in 96 well-plate and 6 well-plate formats at ⁇ 4 x 10 4 cells/well and -1.5 x 10 5 cells/well (respectively).
  • the cells were seeded in MatrigelTM-coated wells in medium consisting of DMEM:F12 containing 20 ng/mL FGF2 and penicillin/streptomycin until use.
  • medium consisting of DMEM:F12 containing 20 ng/mL FGF2 and penicillin/streptomycin until use.
  • the medium was removed and pure water added containing Hoechst 33342 and propidium iodide (each at ⁇ 1 mg/mL; -200 DL was added per well for the 96 well-plate format and ⁇ 2 mL for the 6 well-plate format).
  • the cells were then assessed by light and fluorescent microscopy using a CKX41 microscope at the following intervals, with the number of live cells (only Hoechst stained) and dead cells (both Hoechst and propidium iodide stained) counted: 0, 2, 3 and 5 minutes, then 5 minute intervals until 60 minutes after initial exposure to water. Light and fluorescence images were taken at various time points. The results are shown in Fig. 3.
  • RNA-seq high-resolution gene expression profiling was used to define the genes expressed by human lens cells purified by MACs on the basis of ROR1 expression as described above using ROR1+ cells purified from differentiating CA1 human embryonic stem cell cultures. Briefly, three samples of stranded total RNA from two biological replicates of ROR1 -purified human lens cells were run on one lane of 2x100 paired-end sequencing. The resulting data was mapped and aligned to the genome using the TopHat aligner (Centre for Computational Biology, Johns Hopkins
  • the expression of known lens genes was specifically assessed.
  • two lens gene sets were used: (1) a manually curated lens gene set based on Table 2 in Lachke et al., 2012 (Investigative Ophthalmology and Visual Science 53(3): 1617-27); and (2) a gene set based on the most highly lens-enriched gene in embryonic mouse lens based on iSyTE (Lachke et al., 2012).
  • the mouse embryonic lens gene set is the union of the top 100 highly ranked lens-specific genes from three mouse embryonic days (El 0.5- E12.5), and shows good correlation with the purified lens cells (Fig.4).
  • RNA-seq including key lens genes such as PAX6, PROXl, CRYAA, CRYAB, MAF, FOXE3, SIX3, PITX3, etc.
  • key lens genes such as PAX6, PROXl, CRYAA, CRYAB, MAF, FOXE3, SIX3, PITX3, etc.
  • non-lens cell genes werenot expressed within the 3 RNA-seq including: pluripotent cell genes (e.g., NANOG, OCT4, ZIC3, ESRRB, etc.); endodermal cell genes (e.g., GATA4, GDF3, Von Willebrand factor, etc.);
  • mesodermal cell genes e.g., Brachyury, Goosecoid, CD34, CXCR3, etc.
  • non-lens ectodermal cell genes e.g., RPE65, NEUROD1, Choline acetyl transferase, etc.
  • RNA-seq profiles are a good representative of human lens epithelial cells
  • the genome- wide expression profiles of the three RNA-seq profiles were compared against published microarray data from human lens epithelial cells and human lens fibre cells (Gene Expression Omnibus dataset GSE2256, Hawse JR et al., 2005).
  • the normalised data from this dataset was log2 transformed, and the gene expression levels represented as the mean of the log2 expression of their respective biological triplicates. All three purified human lens cell RNA-seq samples were shown to have good positive correlation with the primary lens epithelial and lens fibre cell profiles, with slightly higher correlation with the epithelial cell profile.
  • RNAseq-microarray correlation is stronger at higher gene expression, consistent with i) the expectation that microarrays have lower resolution at the lower end of gene expression spectrum (i.e., the RNA-seq data better resolves lowly-expressed from non-expressed genes), and ii) the observation of a bimodal distribution of FPKM values for gene expression.
  • the purification of LECs in accordance with the invention provides for downstream application of the purified cells, such as the study of mechanisms of primary and secondary cataract development, the development of primary and secondary cataract drug-screening assays (which require LEC populations essentially free of contaminating non-lens cells and LFCs), and for toxicology assays.
  • Key to the exemplified purification of LECs was the identification of cell surface proteins ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 which appear to be specifically expressed in the lens epithelium at a particular stage of embryonic development.
  • MACS or other cell separation technology
  • LECs Limited access to human lens cells has severely inhibited the development of needed new presbyopia and cataract treatments.
  • the method for purification of LECs as herein described provides a long-awaited source of normal or diseased lens cells to aid the development of new accommodation-retaining, presbyopia and cataract treatments suitable for both adults and children and so has broad clinical, research and commercial applications.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Neurosurgery (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Neurology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Ophthalmology & Optometry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

There is provide a method for the purification of eye lens cells and/or progenitor cells thereof from a mixed population of cells, comprising isolating cells on the basis of outer cell membrane expression of at least one marker selected from the group consisting of ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 from the mixed cell population, and collecting the isolated cells. There are also provided purified preparations of cells expressing one or more of cell surface markers ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2, and progeny of the purified cells, as well as lentoids and lens-like structures prepared from the purified cells or progeny thereof and methods for the provision of the lentoids and lens-like structures.

Description

A METHOD FOR THE PURIFICATION OF EYE LENS CELLS
FIELD OF THE INVENTION
The present invention relates to a method for purifying a population of cells, and to preparations of the cells and uses of them.
BACKGROUND OF THE INVENTION
Hardening of the ocular lens (presbyopia) affects people from approximately age 45. This results in reduced ability to change focus between near and far objects (i.e., reduced accommodation), and affects many aspects of daily life including reading and driving. Between 500 million and 1 billion people worldwide suffer from presbyopia. Current treatments typically consist of corrective spectacles or contact lenses, particularly for close vision.
Due to patient demand, multi-focal contact lenses have been developed that provide some vision correction for people with presbyopia. However, these contact lenses are not available to all patients due to incompatible prescription type or too large pupil size. Moreover, treated patients often discontinue contact lens use due to inherent glare and halos that affect daily activities, as well as discomfort, dryness, infection, ongoing costs and daily handling requirements.
Presbyopia can also be a precursor to loss of lens transparency that leads to blindness (i.e., cataract). Cataract-induced blindness affects 100 million people globally and currently can only be treated surgically. Cataract surgery involves removal of the non-transparent biological lens and implantation of an intraocular lens (IOL). In all cases cataract surgery leads to loss of accommodation, necessitating the use of approaches to try and redress this complication such as multifocal spectacles or IOLs. In Australia alone more than 180,000 cataract operations are performed annually costing in excess of $326 million. Patient numbers and costs are each 10 to 20 times higher in the United States, Europe and Asia. Whilst advancing age is a key risk factor for cataract, other cataracts that occur in children or youths (such as congenital or traumatic paediatric cataract) can result in significant life-long learning, behavioural and psychological issues for the sufferer. This is particularly true in developing countries where paediatric lens removal can occur without optical correction (aphakia) due to difficulties surgeons face choosing IOLs for infant eyes that will grow significantly in the first years of life (i.e., growth that is not easily matched by fixed size, non-accommodating IOLs).
In addition to loss of accommodation consequent to all adult and paediatric cataract surgeries, secondary cataract (or posterior capsule opacification, PCO) is a common complication of primary cataract surgery. PCO results from abnormal growth of residual lens epithelial cells not removed during primary cataract surgery, and further (laser) treatment is required to restore vision in patients who develop PCO. Additional side-effects of paediatric cataract surgery include 'lazy eye' (stabismus), involuntary eye movements (nystagmus), glaucoma, changing ocular optics during patient growth, and slowed behavioural and social development.
Due to population aging the number of cataract patients is expected to double by 2024. New therapies are thus needed to improve our ability to cost-effectively treat primary adult and paediatric cataract, avoid key complications such as secondary cataract and loss of accommodation, and also better treat the lifestyle-altering effects of presbyopia. Critical to this is having access to purified populations of eye lens cells from human and animal sources for use in screening for anti-primary and anti- secondary cataract drugs, development of lens cell toxicology assays and development of new treatments for presbyopia and cataract. Limited access to sufficient numbers of purified lens cells has severely hampered the development of these much needed new drugs and treatments.
The ability of human pluripotent stem cells (hPSCs) to self-renew and differentiate into cells from the three embryonic germ layers highlights them as a promising source of cells for basic research, toxicology screening and drug discovery programs. For the lens of the eye, a critical step towards the treatment of presbyopia and cataract is the development of a scalable human lens cell culture system. Recently, a 3-stage growth factor treatment protocol has been reported that provides for the differentiation of hPSCs into heterogenous cell cultures that contain lens epithelial progenitor-like cells and rudimentary 3-dimensional lens-like structures known as 'lentoids' (Yang et al 2010). Lentoids comprise of a mixture of lens fibre cells (LFCs) and lens epithelial cells (LECs) and do not correctly mirror the complex architecture of the lens, namely, a monolayer of anterior epithelial cells overlying a compacted mass of parallel-aligned, elongated lens fibre cells. Non-lens cells are also produced via this method, and any remaining undifferentiated hPSCs have the potential to generate teratomas and may interfere with the outcome of in vitro differentiation protocols.
Fluorescence activated cell sorting (FACS) can be employed to deplete
"contaminating" cells, and the application of FACS to the purification of hPSC derived LECs based on assessment of published lens cell surface proteins has recently been reported (Mengarelli et al, 2013). However, this process is complex in nature requiring cells cultured for up to 30 days to undergo multiple positive and negative sorts based on the cell surface expression of known lens proteins. This includes positive selection of lens cells that expressed c-Met and/or CD44, with simultaneous depletion of cells that expressed p75, HNK-1, and CD15. Few lens cells are obtained through this process and, due to the large amount of time, complex equipment and expert technical support required for this lens cell differentiation and purification method, the technique is not scalable for future high-throughput applications.
A need remains for a process that enables the provision of large numbers of human LECs that are essentially free of contaminating cells and which can be maintained in culture and directed to form LFCs in a controlled manner, and which is relatively rapid, inexpensive and scalable.
SUMMARY OF THE INVENTION The present invention relates to the identification of cell surface markers that are expressed on the outer cell membrane of particular eye lens cells whilst either being not expressed or simultaneously only relatively lowly expressed on other unwanted tissues or cell types by comparison, and embodiments as described herein can provide for the relatively rapid purification of large numbers of the lens cells.
In particular, in one aspect of the invention there is provided a method for the purification of eye lens cells and/or progenitor cells thereof from a mixed population of cells, comprising isolating cells on the basis of outer cell membrane expression of at least one marker selected from the group consisting of ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 from the mixed cell population, and collecting the isolated cells.
The mixed population of cells can, for example, comprise a mixed population of embryonic cells, postnatal, juvenile or adult cells.
In at least some embodiments, the method may further comprise providing the mixed population of cells by inducing pluripotent stem cells to differentiate into the lens cells and/or progenitor cells thereof.
In one embodiment, the selected marker is RORl .
In another embodiment, the selected marker is GPR161.
In another embodiment, the selected marker is CD81.
In another embodiment, the selected marker is ODZ3.
In another embodiment, the selected marker is SLC7A11.
In another embodiment, the selected marker is SLC16A1.
In another embodiment, the selected marker is SLC23A2.
In at least some embodiments, the marker is selected from RORl, and GPR161.
Accordingly, in another aspect of the invention there is provided a method for the purification of eye lens cells and/or progenitor cells thereof from a mixed population of cells, comprising isolating cells on the basis of outer cell membrane expression of RORl and/or GPR161 from the mixed cell population, and collecting the isolated cells.
Typically, the eye lens cells and/or progenitor cells thereof express the selected marker and one or more other of the markers from the group consisting of RORl, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2. Usually, the eye lens cells or progenitor cells thereof express a majority of the markers and most typically, all of the markers. In one embodiment of the invention the purified eye lens cells are LECs and/or progenitor forms of LECs.
In another embodiment the purified eye lens cells can comprise LECs and/or LFCs, and/or progenitor forms of LECs and/or LFCs. In the instance the purified cells comprise LFCs or progenitor forms of LFCs, the LFCs or progenitor cells thereof may comprise or consist of immature LFCs and/or cells that are in the process of
differentiating from LECs into LFCs.
Typically, the cells purified in accordance with a method embodied by the invention will consist of purified populations of LECs, progenitor forms of LECs and/or early differentiating LECs. Typically, LECs and/or progenitor forms of LECs purified in accordance with a method embodied by the invention express at least RORl from the group of cell surface markers.
Typically, LFCs and/or progenitor forms of LECs purified in accordance with the invention express at least GPR161 from the group of the cell surface markers.
Typically, the isolation of the cells comprises contacting cells in the mixed cell population with a respective binding agent for binding to the selected marker and separating the cells to which the binding agent has bound from the mixed population. The binding agent can be any suitable ligand for the selected marker, or for instance, an antibody or a binding fragment thereof. Whilst the eye lens cells and/or progenitor cells thereof can be purified from the mixed population of cells utilising a binding agent for a single one of the cell surface markers (e.g., RORl) in at least some embodiments of the invention a plurality of binding agents may be utilised, each of the binding agents binding to a different one of the cell surface markers, respectively. For example, one binding agent which binds to RORl and another binding agent which binds to GPR161 (and so on) can be employed.
In at least some embodiments of the purification method of the invention the cells bound by the respective binding agent are separated from the mixed population of cells by flow cytometry or magnetic separation. Any suitable magnetic separation protocol may be employed but typically, the magnetic separation comprises magnetic activated cell sorting (MACS).
Although a combination of cell separation techniques may be utilised to provide a purified population of cells in accordance with the invention, magnetic separation alone is typically used to separate the cells from the mixed population of cells.
In another aspect there is provided a preparation of eye lens cells purified by a method of the invention.
In another aspect of the invention there is provided a purified population of eye lens cells and/or progenitor cells thereof, having outer cell membrane expression of at least one marker selected from the group consisting of RORl, GPR161, CD81, ODZ3, SLC7A11, SCL16A1 and SLC23A2.
There is also further provided progeny cells from cell preparations embodied by the invention. In another aspect there is provided an assay for screening the effect of a test agent on eye lens cells, comprising:
providing a purified preparation of eye lens cells and/or progenitor cells thereof in accordance with an embodiment of the invention, or progeny cells thereof;
treating the cells with at least one test agent;
culturing the treated cells for a predetermined period under suitable conditions for maintenance of the cells; and
assessing the effect of the test agent on the cultured cells.
In one embodiment of the invention the assay is a toxicology assay. In another embodiment the assay is a drug screening assay for assessing the effect of a therapeutic or putative drug on the cells.
The cells purified or utilised in accordance with a method embodied by the invention can be normal cells, aberrant cells (e.g., diseased cells), or mixtures of the foregoing.
In still another aspect there is provided a method for providing a lentoid, comprising culturing purified cells embodied by the invention under conditions suitable for the generation of the lentoid and for a period of time to generate the lentoid. The method can also comprise removing the lentoid from the cultured cells for further culturing and/or study.
In yet another aspect there is provided a lentoid provided by a method of the invention.
Advantageously, embodiments of a purification method as described herein may provide for the preparation of large numbers of LECs or LFCs (particularly embryonic LECs or LFCs) in high purity (e.g., pure or substantially pure cell preparations) which can be subsequently utilised in drug screening and toxicology assays, or for being cultured for research or other purposes. Further, at least some forms of the invention provide for the purification of the cells rapidly, inexpensively and without the need for access to complex equipment such as flow cytometers. In particular, methods for purification of the cells may be carried out using common, readily available laboratory equipment. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the invention as it existed in Australia or elsewhere before the priority date of this application.
The features and advantages of the invention will become further apparent from the following detailed description of non-limiting embodiments thereof together with the accompanying drawings.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1 : A) GenePaint analysis of gene lists (generated using the Excel-based macro) identified ROR1 (shown), GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 (not shown) as having highly-restricted lens expression patterns in mouse embryos approximately E14.5. B) Flow cytometry analysis shows that performing MACS using commercially-available antibodies against ROR1 enables lens cell purification from impure cell populations (in this case, differentiating CA1 human embryonic stem cells). C) Real-time PCR analysis shows that (MACS-) purified human lens cells possess increased transcript levels for lens epithelial cell biomarkers (e.g., a-crystallin) and lower levels of lens fibre cell biomarkers (e.g., β-crystallin) compared to (MACS-) non-expressing ('negative') cells and an impure starting cell population ('Day 18 pre-MACS').
Figure 2: A) A light micrograph showing MACS-purified lens cells in culture (derived from differentiating CA1 human embryonic stem cell cultures; lOx
magnification). B-E) Culture of (MACS-) purified lens cells enables lens drug and toxicology screening (blue = Hoechst nuclear stain for total cells; red = propidium iodide staining for dead cells): control-treated cells retain high cell numbers (B) with little cell death (C), whereas hydrogen peroxide (H202)-treatment (D, E) results in massive cell death (E) leaving only residual dead cells (E). F) Quantification of the effects of H202-treatment on purified lens cells show the effects of H202 are highly significant.
Figure 3: Graph showing survival of hLECs purified in accordance with an embodiment of the invention exposed to pure water in a cell death assay. Figure 4: Correlation analysis showing the gene expression profiles of purified human lens cell samples are more similar to early embryonic mouse stages than later embryonic stages or postnatal stages.
Figure 5: Expression of selected key lens genes by purified human lens cells. (A, B) histograms of gene expression for Cufflinks FPKM and HT-seq counts.
Figure 6: Expression of key lens genes by purified human lens cells. (A,B) histogram of gene expression for Cufflinks FPKM and HT-seq counts, respectively.
Figure 7: A-G) El 4.5 mouse in situ hybridisation data showing lens specificity of 7 cell membrane proteins for human lens cell purification that are expressed by adult primary human lens epithelial cells. H) RNA-Seq expression levels show all of lens cell purification markers ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 are expressed in ROR1 -purified human lens epithelial cells..
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION
Whilst the mixed population of cells utilised in a purification method embodied by the invention is preferably a population of differentiated (or differentiating) stem cells (e.g., pluripotent stem cells), the cells may be obtained from various other sources including adult cells, cultured or non-cultured primary lens cells, lens cell lines, and for instance, transdifferentiation of iris or corneal cells (e.g., newts, frogs and other sources). Primary lens cells, for example, can be obtained directly from isolated eye lens tissue and subjected to a method as described herein. Pluripotent stem cells which may be induced to differentiate into lens cells or progenitor forms thereof purified in accordance with the invention include WA01 (HI) human embryonic stem cells
(WiCell Research Institute, Madison, WI, USA), CA1 human embryonic stem cells (Adewumi et al., 2007), MEL1 human embryonic stem cells (Kolle et al., 2009), iPS(201B2) human induced pluripotent stem cells (Takahashi et al., 2007), or other suitable stem cells.
ROR1 (Receptor Tyrosine Kinase-like Orphan Receptor 1) has been reported to modulate neurite growth in the central nervous system, and e.g., human and mouse nucleic acid sequences are available on publicly accessible databases (e.g., Human: Uniprot ID. Q001083592, NM_001083592 (mRNA), NP 001077061 (AA); Mouse: Uniprot ID. Q9Z139, NM_013845 (mRNA), NP_038873 (AA)). GPR161 is a previously reported G-protein coupled receptor (e.g., Human: Uniprot ID. Q8N6U8; NM_001267609 (mRNA), NP_00125438 (AA); Mouse: Uniprot ID. B2RPY5,
NM_001081126 (mRNA), NP_0010745959 (AA)). CD81 has been reported to complex with integrins and to appear to promote muscle cell fusion and support myotube maintenance, and may be involved in signal transduction (e.g., Human:
Uniprot ID. P60033, NM_001297649 (mRNA), NP_001284578 (AA); Mouse: Uniprot ID. P35762, NM_ 133655 (mRNA), NP_598416 (AA)). SLC7A11 has been reported to be an anionic amino acid antiporter that is highly specific for cysteine and glutamate (e.g., Human: Uniprot ID. Q9UPY5, NM_014331 (mRNA), NP_ 055146 (AA); Mouse: Uniprot ID. Q9WTR6, NM 011990 (mRNA), NP 036120 (AA)). ODZ3 belongs to the tenascin family and has been reported to possibly be a cell signal transducer (e.g., Human: Uniprot ID. Q9UKZ4, NM_001163278 (mRNA), NP_001156750 (AA);
Mouse: Uniprot ID. Q9WTS4, NM_011855 (mRNA), NP_035985 (AA)). SLC16A1 has been reported to be a proton-linked monocarboxylate transporter for transport of monocarboxylates such as lactate and pyruvate across the plasma membrane (e.g., Human: Uniprot ID. P53985, NM_ 001166496 (mRNA), NP 001159968 (AA); Mouse: Uniprot ID. P53986, NM_ 009196 (mRNA), NP_033222 (AA)). SLC23A2 has been reported to be a sodium-dependent ascorbic acid (vitamin C) transporter (e.g., Human: Uniprot ID. Q9UGH3, NM 005116 (mRNA), NP_ 005107 (AA); Mouse: Uniprot ID. Q9EPR4, NM_018824 (mRNA), NP_061294 (AA)). Purification of the lens cells and/or progenitor cells thereof on the basis of homologous, variant and mutant forms of the selected cell surface marker(s) is expressly encompassed by the present invention Cells that may be purified in accordance with a method of the invention include LECs, progenitors of LECs and early-differentiating LFCs (e.g., expressing one or more of ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2).. The term "early-differentiating" as used herein encompasses immature LFCs, and the term "progenitor" as used herein encompasses precursor cells that give rise to LECs and/or LFCs. For example, lens progenitor cells within the preplacodal region of late gastrulation stage embryos.
When embryonic cells are purified in accordance with the invention the cells will typically be LECs, early-differentiating LFCs or progenitor cells thereof.
Typically, the embryonic cells will be at least equivalent to lens progenitor cells in the preplacodal region of gastrula stage embryos, or later stage lens progenitor and lens cells.
Various purification techniques may be utilised to purify lens cells expressing the selected cell surface marker(s) in accordance with the invention including, but not limited to, FACS and magnetic separation techniques. For separation by FACS, the mixed cell population may be incubated with a respective primary antibody specific for the selected marker prior to washing and incubating the cells with a secondary antibody specific for binding to the primary antibody and which is labelled with a fluorescent marker (e.g., phycoerythrin or FITC) for separation of the labelled cells utilising a flow cytometer employing known protocols.
Magnetic separation techniques that may be employed in a method of the invention can utilise paramagnetic substrate particles coated with a respective antibody or other binding agent for binding either directly or indirectly to the selected cell surface marker(s) expressed on the target cell(s). Indirect binding may, for example, be achieved by the binding of a secondary antibody on the magnetic particle to a primary antibody bound to the selected marker(s) expressed on the outer membrane of the target cell. Alternatively, the magnetic particle may be coupled to the target cell via streptavidin provided on the particle linking to a biotinylated primary antibody or other binding agent bound to the selected marker(s) expressed on the cell, and all such variations are expressly encompassed. To isolate the magnetic particle-bound cells from the mixed cell population, the cells are placed in a magnetic field provided by fixed magnet(s) to which the magnetic particles are attracted allowing those cells not bound by the magnetic particles to be e.g., eluted, decanted, or otherwise physically separated from the magnetically bound cells.
Other cell separation techniques involving binding of target lens cells expressing the selected cell surface marker(s) to a solid substrate for removal of non-bound cells may also be utilised. Suitable such solid substrates that may be used include plastics (e.g., polypropylene and other suitable plastics) and synthetic resins, and beads of e.g., latex, polystyrene, dextran, agarose, sepharose, glass and synthetic resins as described above. An antibody, binding fragment or other binding agent as described herein can be bound to a solid substrate covalently utilizing commonly employed amide or ester linkers or, for instance, by adsorption. Protocols for the preparation of such solid substrates for affinity separation techniques are for instance described in Current Protocols in Molecular Biology - Ausubel FM. et al, Wiley-Interscience, 1988 and subsequent updates thereof.
Moreover, collected target cells may be washed one or more times with a suitable physiologically acceptable buffer and subjected to additional round(s) of separation in order to further increase the purity of the isolated target lens cells.
Likewise, a combination of cell purification techniques as described herein may be utilised (e.g., one or more rounds of magnetic separation followed by FACS).
Generally though, the use of magnetic separation (e.g., MACS) will be sufficient to provide a highly purified population of the target lens cells. Magnetic separation techniques that may be employed include Dynabead™ and MACS techniques. MACS purification has the advantage over Dynabead™ cell purification in that commercially available magnetic nanoparticles used in MACS are biodegradable, and so there is no need for use of a releasing agent to separate the magnetic beads from the captured lens cells.
Typically, the binding agent used in a method of the invention will be an antibody. The antibody can be polyclonal or monoclonal although the latter is preferred. The production of polyclonal antibodies and monoclonal antibodies is well established in the art (e.g., see Antibodies, A Laboratory Manual. Harlow & Lane Eds. Cold Spring Harbour Press, 1988). For polyclonal antibodies, a mammal such as a sheep, goat or rat is immunized with an antigenic fragment of the target protein (e.g., human ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 or SLC23A2) and anti- sera is subsequently isolated from the mammal prior to purification of the antibodies generated against the antigen by standard affinity chromatography techniques such as Sepharose-Protein A chromatography. Desirably, the mammal is periodically challenged with the relevant antigen to establish and/or maintain high antibody titer. To produce monoclonal antibodies, B lymphocytes can be isolated from the immunized mammal and fused with immortalizing cells (e.g., myeloma cells) using somatic cell fusion techniques (e.g., employing polyethylene glycol) to produce hybridoma cells (e.g., see Handbook of Experimental Immunology, Weir et al Eds. Blackwell Scientific Publications. 4th Ed. 1986). The selection of hybrid cells may be achieved by culturing cells in hypoxanthine-aminopterin-thymidine (HAT) medium, and selected hybridoma cells then screened for production of antibodies specific for the target protein by enzyme linked immunosorbant assay (ELISA) or other immunoassay. However, it will be understood that any suitable commercially available such antibody can be utilised in a method embodied by the invention.
Antibodies that may be utilised for binding to ROR1 for the purification of lens cells or progenitor cells thereof in accordance with the invention include AF2000 (RandD Systems, Inc., Minneapolis, MN 55414, USA). Antibodies for binding to GP161 may for instance be selected from ab58679 (Abeam, Cambridge, MA 02139- 1517, USA), ΑΒΓΝ1734983 (Antibodies Online Inc., Atlanta, GA 30338, USA), orb 157327 (Biorbyt LLC, San Francisco, CA 94104, USA), MC-331 (MBL
International Corp, Woburn, MA 01801, USA), TA316812 (Origene, Inc., Rockville, MD 20850, USA), PA5-33652 (Pierce Antibodies, Thermo Scientific Inc., Rockford, IL 61101, USA), and sc-101966 (Santa Cruz, Inc., Dallas, TX 75220, USA). Antibodies for binding to CD81 may for instance be selected from AP6631 (Abgent, Inc., San Diego, CA 92124, USA), orb36798 (Biorbyt LLC, San Francisco, CA 94104, USA), OAAB03662 (Aviva Systems Biology Corp., San Diego, CA 92121, USA), GTX81801 (GeneTex Inc., Irvine, CA 92606, USA), and PA5-1358 (Thermo Fisher Scientific Inc., Rockford, IL 61101, USA). Antibodies for binding to ODZ3 include orbl58049 (Biorbyt LLC, San Francisco, CA 94104, USA), TA321274 (CliniSciences, Nanterre 92000, FR), and sc-136920 (Santa Cruz, Inc., Dallas, TX 75220, USA). Antibodies for binding to SLC7A11 may for instance be selected from antibodies abl 11822, ab60171, ab99059, abl 12403, and ab84171 (Abeam, Cambridge, MA 02139-1517, USA),
12691S (Cell Signalling, Inc., Danvers, MA 01923, USA), and sc-79360 (Santa Cruz, Inc., Dallas, TX 75220, USA). Antibodies for binding to SLC16A1 may for instance be selected from ab85021 and ab90582 (Abeam, Cambridge, MA 02139-1517, USA), LS- C335287 (LSBio, Inc., Seattle, WA 98121, USA), LS-C341521 (LSBio, Inc., Seattle, WA 98121, USA), and HPA003324-100UL (Sigma-Aldrich, LLC, St. Louis, MO 63103, USA). Antibodies for binding to SLC23A2 include antibodies sc-9927, sc-30113, and sc-376090 (Santa Cruz, Inc., Dallas, TX 75220, USA).
Other binding agents which may be used in a method of the invention besides whole antibodies include binding fragments of antibodies and other proteinaceous agents that bind to the selected surface marker(s), and streptavidin, biotin and the like. The term "binding fragment" of an antibody expressly includes within its scope Fab and (Fab')2 fragments obtainable by papain or pepsin proteolytic cleavage respectively, variable domains of antibodies (e.g., Fv fragments), and antibody single chain variable fragments (scFvs) and multimer forms thereof such as bivalent scFvs (e.g., bivalent and diabodies), trivalent scFvs (triabodies) and tetravalent scFvs (tetrabodies), that bind to the target protein. Strategies for identifying other proteinaceous agents that may be used in a method for the purification of lens cells as described herein include large scale screening techniques. For instance, phage display library protocols provide an efficient way of testing a vast number of potential peptide binding agents.
Phage display libraries express random transgenic peptides or antibody variable domain(s) of known length on the surface of the selected bacteriophage. Each phage clone displays a distinct such peptide sequence. The peptide sequences are fused with major or minor coat proteins of the selected phage type and can be produced by inserting random oligonucleotides in DNA encoding the coat protein, transfecting the resulting construct into a suitable host bacterial strain, and generating phage particles upon superinfection of the bacterial strain with helper phage. Peptides which bind to the selected cell surface marker(s) can, for instance, be identified by contacting lens cells expressing the target protein to identify phage clones in the library which bind to the protein. Unbound phage is washed away and the remaining bound phage is recovered. The pool of bound phage can be enriched by subjecting the bound phage to a number of such biopanning cycles, wherein the bound phage is collected and amplified utilising suitable host bacteria before being subjected to the next cycle. The sequence of the binding peptide of an isolated phage clone may then be identified by sequencing the relevant coat protein of the clone, and comparing that sequence with the known sequence for the native phage coat protein.
Drug screening and toxicology assays in accordance with the invention will typically involve culturing cells purified as described herein in the presence of a known or putative drug or toxicant for a period of time sufficient for the effects of the drug or toxicant to be observed directly (e.g., visually or with the aid of a microscope) or otherwise be detectable such as by staining of the cells or subjecting the cells or cell culture supernatant to appropriate analysis. The cells can be cultured with the drug or toxicant alone or in combination with one or more other agents (e.g., cytokines, cell regulatory agents, cell differentiation agents, growth factors etc.) and/or cell types. The cells may be cultured in such assays for e.g., minutes, hours, overnight or days, and may require the culture medium to be changed, refreshed or supplemented during the culture period. The cells utilised in toxicity screening or drug testing assays as described herein can be normal cells or aberrant cells purified in accordance with a method embodied by the invention. Such aberrant cells can, for example, be cells that are deficient in one or more characteristics compared to normal cells or be diseased cells (e.g., cancer cells, abnormally growing LECs, cells which causing primary or secondary cataract, cells affected by age-related cataract, and cells with congenital or acquired mutation(s) that cause cataract, etc). For example, an assay as described herein may be utilised for screening for an anti-posterior capsule opacification (PCO) drug. PCO arises from residual primary human LECs that are not removed during primary cataract surgery. These residual lens epithelial cells migrate along the interior surface of the lens capsular bag (that holds the intraocular lens implanted during cataract surgery). Once the residual primary LECs reach the posterior of the capsular bag the local growth factor environment causes the cells to elongate and wrinkle the capsule which causes light scatter (i.e., PCO). Once PCO occurs vision can only currently be restored by laser- based cutting of the posterior lens capsule (which then falls away from the capsular bag but remains in the eye). Thus, for example, an assay as described herein may be utilised to identify one or more compounds that kill, inhibit the migration of, render mitotically inactive, or differentiate to LFCs residual LECs left after cataract surgery..
For toxicology screening or drug testing assays as described herein utilising cells purified in accordance with an embodiment of the invention, the cells may be cultured in the wells of a tissue culture plate (e.g. a 12, 24 or 96 well or other plate or dish) and exposed to the test agent for a predetermined period of time prior to assessing the effect of the test agent on the cells. Typically, the cells will be cultured at a density in a range of from tens of cells per well (e.g., for 96, 384, 1536 well plates) up to hundreds of thousands of cells per well or dish (e.g., for multi-well plates). Suitable culture medium for culturing the cells may be selected from typical base media (e.g., RPMI, DMEM, DMEM, F12, F12, Ml 99, etc) with additional growth supplements (e.g., fetal bovine serum, bFGF, ocular fluids such as aqueous fluid or vitreous fluid, etc) with or without antibiotics and/or anti-fungal agents.
The cells may be cultured in the presence of the test agent for any predetermined appropriate time in order for the effect(s) of the test agent(s) to be observed such as from 1 minute to 1 or more hours (e.g., 2, 3, 4, 5, 6, 7 or 8 hours, overnight, 1 day, 2 days etc.) depending on the nature and concentration of the test agent. If appropriate, fresh culture media can be added to the test wells or media containing the test agent can be aspirated from the wells and replaced with fresh media with or without the test agent.
The effect of the test agent on the cells can be evaluated by any manner that quantifies the loss of live cells and/or the appearance of dead cells such as by MTT growth assay, detection of live and dead cell numbers such as by involving propidium iodide staining of viable cells or use of other cell viability dyes or stains (e.g., nigrosin, LIVE/DEAD® Cell Viability Assay reagents, etc), visual evaluation of cell
morphology, [ H] -thymidine uptake and other means for evaluating cellular expression, in-situ hybridisation methods, radioimmunoassays, methods for the evaluation of gene expression such as by polymerase chain reaction (PC ) techniques, etc.
Test agents that may be assessed by a method as described herein utilising purified cells embodied by the invention include putative anti- primary or secondary cataract drugs as well as inhibitors or agonists of cell signalling pathway (e.g., kinases, receptors etc.), ion channels, enzymes, and proteasome and proteasome pathway members, etc.
Purified cells embodied by the invention (e.g., LECs, early-differentiating LFCs and progenitor forms thereof) may also be cultured under suitable conditions to generate lentoid(s). This may involve culturing the cells at cell densities sufficient for the formation of a lentoid, and (for example) supplementing culture media with one or more cell differentiating agents (e.g., FGF family members such as bFGF, and/or other growth factors, ocular fluids such as vitreous fluid, etc) for promoting the formation of the lentoid. The term "lentoid" in the context of the present invention is to be taken to encompass a 3 -dimensional lens like structure of tissue(s) comprising populations of cells selected from LECs, LFCs, early differentiating LFCs, and progenitor cell forms of the aforementioned cells.
In particular, a lentoid may be prepared by culturing purified cells embodied by the invention at a cell concentration and in the presence of bFGF sufficient for promoting the development of the lentoid as described further below. Typically, the cells will be cultured until confluent. The cell culture medium will generally contain bFGF at a concentration of at least 1 ng/ml, more usually at least 20 ng/ml and most preferably in a range of from 50 ng/ml to about 100 ng/mL. The culture medium utilised may, for example be selected from RPMI, DMEM, DMEM:F12, F12, Ml 99, etc. Conventional methods for the production of a lentoid in which cells purified in accordance with the present invention can be utilised are described in, Yang et al, 2010, the entire contents of which is incorporated herein in its entirety by cross reference, and for research into the generation of lenses or lens-like structures. In particular, cells purified in accordance with the invention have application to the in vitro generation of lenses and lens-like structures via methods as described in e.g., O'Connor and McAvoy, 2007, the entire contents of which is also incorporated herein in its entirety by cross- reference.
The lens cells may be purified in accordance with the invention to high levels of purity for use in drug screening and toxicology assays or for other uses such as described herein. The term "purified" in the context of the invention is to be taken to encompass populations of the target lens cell(s) that are of a higher purity than the mixed cell population from which they were isolated. Purified preparations of the lens cells expressing the selected cell surface marker(s) (e.g., ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and/or SLC23A2) include preparations that have a purity level of at least about e.g., 80%, 85%, 90% or 95% or greater (e.g., 96%, 97%, 98%, 99% or 100%). By "essentially" or "substantially" pure or purified is meant that the cell preparation of the invention is pure or may contain a "contaminating" proportion of cells e.g., non-RORl (i.e., ROR1"), non-GPR161 (i.e., GPR161") , non-CD81 (i.e., CD81"), non-ODZ3 (i.e., ODZ3"), non-SLC7Al 1 (i.e., SLC7A11"), non-SLC16Al (i.e., SLC16A1") and/or non-SLC23A2 (i.e., SLC23A2") expressing cells. For example, dead cells captured non-specifically during MACS. Such contaminating cells may comprise e.g., <10% of the cell preparation (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% or less) depending on the conditions the cells experience.
The cells purified in accordance with a method embodied by the present invention may be obtained from various animals including but not limited to birds, amphibians (e.g., frogs and newts), and mammals such as rabbits and members of the rodent (e.g., mice, rats and hamsters), bovine, ovine, equine, porcine, canine, feline and primate (e.g., chimpanzees, Rhesus monkeys and baboons) families, and humans.
Typically, the cells will be human cells (e.g., primary human eye lens cells, embryonic cells, etc). In particularly preferred embodiments the cells are cells that are obtained by inducing human pluripotent stems cells to differentiate into lens cells, and may include or comprise differentiated lens cells and/or precursor forms thereof in the lens cell lineage that express the selected cell surface marker(s) i.e., RORl, GPR161, CD81, 0DZ3, SLC7A11, SLC16A1 and/or SLC23A2).
The invention is further described below with reference to a number of non- limiting Examples.
EXAMPLE 1 Identification of lens cells markers
The 3 stage growth factor protocol of Yang et al, 2010 was assessed for its ability to produce crystallin-expressing human eye lens epithelial cells (LECs) and eye lens fibre cells (LFCs) in culture using CA1 human pluripotent stem cells (hPSCs)
(Adewumi et al, 2007). Protocols for the culturing and differentiation of the cells as per Yang et al, 2010 are set out below.
1. Harvesting and plating human pluripotent stem cells (hPSCs)
Reagents
Human pluripotent cell medium (e.g., mTeSRl, StemCell Technologies)
- DMEM:F12 + 10% FBS
PBS (Phosphate buffered saline)
- TrypLE™ cell dissociation enzyme (Gibco) or Accutase (StemCell
Technologies, Cat. 07920)
15 ml Falcon tube(s)
2 ml, 5 ml, 10 ml sterile plastic pipettes
sterile p20/p200 pipete tips
- 96-well plate
hemacytometer
cell viability stain (e.g. Nigrosin)
60 mm tissue culture dish
- lOOx (1 mM) Rock inhibitor (Ri) stock)
- growth factors (Noggin, FGF2, BMP4, BMP7, Wnt3a)
media supplements (N2, B27) 2. Cell culture protocols
2.1 Incubate hPSCs with Ri @ 10 μΐ^ per mL of media for 1 hr (e.g., final
concentration 10 μΜ)
2.2 Remove medium and add TrypLE™ cell dissociation enzyme (2 ml/60 mm dish, 1 ml/35 mm dish), and incubate for ~7 to 10 mins, at 37°C, 5% C02.
2.3 Gently wash off hPSCs with PBS using 2 ml plastic pipette and transfer to 15 ml tube, then centrifuge cells at 300g for 5 mins.
2.4 Carefully remove supernatant and add 1 ml of DMEM:F12 + 10% FBS. Carry out 2 independent cell counts and calculate cell concentration/ total cell number
(Perform 3 if a significant difference is obtained from the first two). For each cell count: 10 μΐ. of hPSCs + 40 μΐ. of Nigrosin.
2.5 Use cells as required. Plate ~2 x 105 hPSCs per 35 mm dish containing 2 ml each of mTeSRl + Ri to gain confluent cell monolayer after 3-4 days
2.6 Change media next day to remove Ri, then media change every subsequent day until cell differentiation protocol is commenced.
3. Differentiation of hPSCs 3.1 Day 1 to 5: 3 ml every other day (6 mL on weekends)
Figure imgf000019_0001
Day 6 to 17: 3 ml every other day (6 mL on weekends)
Day 6-17 Volume per mL
FGF2 100 ng/ml 1 ul
BMP4 (20 ng/ml) 0.2 ul BMP7 (20 ng/ml) 0.2 ul
DMEMF12 1 mL
Day 18 onwards: 3 ml every other day (6 mL on weekends)
Day 18> Volume per mL
FGF2 100 ng/ml 1 ul
Wnt-3a (20ng/mL) 0.5 ul
DMEMF12 1 mL
Similar expression profiles of the lens specific genes Pax6, aB crystallin and βΒ crystallin as well as comparable numbers of lentoid like structures were found as previously reported by Yang et al, 2010. Expression of aB crystallin was observed to spike between days 10-18 and lentoid growth reached a peak at -day 18 of culture. Taken together, the results indicate that LEC production was at its height by day 18 but that contaminating cells were present.
From the inventors knowledge no LEC (or LFC) specific surface antigen that may be used to simply and efficiently aid the selection of only LECs (or LFCs) from the mixed cell population resulting from the above 3 -stage cell differentiation protocol has been previously identified.
EXAMPLE 2 Development of macro
To identify specific antigen for use in isolating LEC or LFC cells, an Excel- based macro was developed using visual basics that organizes gene expression data based on relative expression levels across multiple samples. The macro was written to perform a primary sort of the rows within 3 columns of the input worksheet in order to rank genes from highest to lowest expression across these 3 samples. Where analysis of greater than 3 samples is required, a secondary sort of the rows in an additional 3 columns can be performed in comparison to the first 3 columns. This enables ranking of additional replicate array data from the same or a different cell type or tissue. To use the macro, user-downloaded data from public gene expression repositories (e.g., the Gene Expression Omnibus, GEO) is condensed into a single Excel spread-sheet; the minimum information required for macro function being a unique gene identifier in alphabetical order (e.g., Affymetrix ID) and an associated expression value (e.g., present and absent calls; expression value; etc.) from each microarray sample of interest.
The GEO dataset GSE2256 (Hawse et al., 2005) was analysed via the macro to identify membrane proteins expressed by LECs. Output gene lists from the macro were then analysed via a pipeline of publically-available tools to identify new lens cell biomarkers, including: gene ontology assessment (via the DAVID webserver, National Institute of Alergy and Infectious Diseases (NIAD), National Cancer Institute,
Frederick, MD 21702, USA), transcriptional regulation (via the PASTAA webserver (Max Planck Institute for Molecular Genetics, Berlin, Germany;
http://trap.molgen.mpg.de/cgi-bin/home.cgi), and embryonic gene expression patterns (e.g., via the GenePaint webserver (Genepaint Org, Max -Planck Institute of Biophysical Chemistry, Goettingen, Germany; http://genepaint.org)).
A list of 22,275 genes was sorted by the macro and further analysis identified cell membrane proteins in LECs and LFCs (e.g., 44 cell surface receptors identified in the LEC group, 15 in the LFC group, and 60 common to both tissues). In situ hybridisation expression patterns in mouse embryos are available via the Genepaint webserver, Max Plank Institute for Biophysical Chemistry, Goettingen, Germany. In situ hybridisation allows all stages of lens development to be examined i.e., lens pit (E10.5), lens vesicle (El 1.5), primary lens fiber cells (E12.5), and differentiating secondary lens fiber cells (E14.5). However, as the culture system used herein described further below best reflects a stage where immature epithelium and
differentiating LFCs are present, stage E14.5 was assessed.
The ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 genes were identified to have gene expression patterns highly restricted to the eye lens during embryonic development. For example, ROR1 showed a strong anterior lens epithelial gene expression with little expression in other tissues (see Fig. 1 and Fig. 6).
Assessment of literature indicated that some of these genes are known to be expressed in the lens, for example, at E14.5, Gprl61 transcripts are expressed in at least differentiating lens fibre cells (Matteson and Desai, 2008); ROR1 has been reported as being expressed in anterior LECs, the nervous system and other tissues such as the in the heart, lung, kidney and thymus, in addition to the brain(Matsuda 2001; Oishi 1999); etc.
The immortalised foetal human lens epithelial cell line FHL124 was assessed for expression of RORl and GPR161 via polymerase chain reaction (PCR), and both transcripts were found to be expressed. Additional assessment of human lens cell differentiation samples at different time points also showed expression of both transcripts. RNA-seq analysis also showed all 7 lens cell purification markers RORl, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 to be expressed in purified human lens cells (Fig. 6), as further described below in Example 4.
EXAMPLE 3 Magnetic purification of lens cells
As RORl expression was found to be restricted to LECs it was used as a marker receptor for magnetic activated cell sorting (MACS). The FHL124 cell line was used in antibody titrations to identify an optimal concentration of RORl antibody using a commercially available antibody. These cells were also used to optimise concentrations of aB and aA crystallin antibodies due to the known expression of these proteins in LECs. Protocols for MACS are set out below. 1. Magnetic-activated cell sorting (MACS) protocol
1.1 Materials
• Cell culture
• Phosphate buffered saline (PBS; -Mg/Ca) with 2% (v/v) BSA (Invitrogen,
14190250)
· 1.5 mL microcentrifuge tubes (Scientific Specialities Inc, 1212-00)
• 40 μηι cell strainer (BD Biosciences, 352340;)
• Haemocytometer
• Trypan Blue (StemCell Technologies, Cat # 07050)
• MS columns (Miltenyi Biotech, 130-042-201, 25 columns, 107 held, in total 2x 108)
• Antibody biotinylation kit (Miltenyi Biotech, Cat # 130-093-385)
• MACS BSA Stock Solution (Miltenyi Biotech, 130-091-376)
• Anti Biotin MicroBeads (1 mL) (Miltenyi Biotech, Cat # 130-090-485) • Human RORl Affinity Purified Polyclonal Ab, (isotype: Goat) (R&D systems, Cat # AF2000)
1.1 Biotinylation of RORl antibody (at least 24 hours prior to MACS protocol).
Briefly, 150 μΐ^ of 0.2 mg/ml RORl antibody was diluted withl50 μΐ^ of lx
PBS. 100 μΐ^ of the mixture was then added to each tube from biotinylation kit as per manufacturer's instructions, and incubated in fume cabinet for 24 hrs prior to storage at 4°C for future use. 2. Preparation for MACS separation of differentiating hPSCs
2.1 Make 0.5 % BSA pH 7.2 in PBS (-Ca/-Mg) and 2 mM EDTA. (Add 2 mL 10% BSA, 160 μΐ. 0.5M EDTA, 37.48 mL PBS (-Ca/-Mg)). Keep buffer on ice or store at 4°C until use
2.2 Incubate cells at 37° with Ri at 30 μΐ^ per 3 mL of media for 1 hr.
2.3 Add TryPLE™ at 2 mL per 35mm dish, and incubate cells at 37°C for 8 min.
Collect the cells off the culture dish using a 2 mL pipette and filter the cell suspension through a 40 μιη cell strainer into a 50 mL tube.
2.4 Gently wash the plate with 2 mL PBS and filter through a 40 μιη cell strainer into a 50 mL tube.
2.5 Transfer the single cell suspension to a 15 mL tube and centrifuge (300g, 5 min)
2.6 Remove the supernatant and resuspend cells in 1 mL of 2% BSA/PBS.
2.7 Take an aliquot for cell count on haemocytometer.
2.8 Remove enough cells for pre-MACS FACS (lxlO5 per sample extracellular antigen, 2xl05 per sample intracellular antigen) follow staining method.
2.9 Remove enough cells for MACS (3xl06 per MS column) and keep on ice.
3. Staining differentiated hPSCs with antibodies 3.1 Resuspend cells in 40 μΐ, of biotinylated RORl antibody per 3 x 106 cells.
3.2 Add 60 μΐ, of MACS buffer pre-cooled in refrigerator.
3.3 Incubated in pre-cooled rack in fridge for 20 min (2-8°C). 3.4 Gently add 1 mL cooled MACS buffer to wash cells and centrifuge (300g, 5 min, 4°C).
3.5 Remove supernatant and resuspend cells in 80 μΐ, of buffer and add 20 μΐ, of anti-biotin microbeads.
3.6 Incubated in pre-cooled rack in fridge for 15 min (2-8°C).
3.7 Gently add 1 mL cooled MACS buffer to wash cells and centrifuge (300g, 5 min, 4°C).
4. Magnetic separation of RORl+ve lens cells from differentiating hPSC
culture
4.1 Place MACS column in magnetic field of the MAC separator according to the number of cells. Make sure column reservoir is empty before moving to the next step.
4.2 Rinse column with 500 μΐ^ of buffer
4.3 Apply cell suspension to the column. Collect flow through containing
unlabelled cells.
4.4 Wash column with 500 μΐ^ of buffer 3x when the column reservoir is empty after each wash. Collect unlabelled cells that pass through with the cells collected in previous step.
4.5 Remove column from the separator and place it on suitable collection tube.
4.6 Pipette 1 mL of buffer firmly to remove the magnetically labeled cells out of the column into the collection tube.
4.7 The collected RORl+ cells can be replated or are ready for downstream
applications such as FACS staining.
Flow cytometry detection of RORl labelled cells determined the total percentage of cells expressing RORl in day 18 cultures prior to MACS to be -60%. Once cells were purified, flow cytometry showed one dominant population of RORl expressing cells with up to -99% of MACS purified cells RORl positive. This is further supported by flow cytometry assessment of positively collected RORl+ cells showing 99% expression of the LEC marker aB-crystallin. PCR analysis of sorted cells showed a higher expression of aB-crystallin in RORl collected cells as opposed to the negative and unsorted fractions. Additionally, PB3-crystallin (a marker of lens fibre cell lineage) was more highly expressed in unsorted and RORl negative cell populations. Together these results show that RORl can be used to positively select LECs from a mixed population of cells. However, antibodies specific for other of the cell surface markers GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 may also, or alternatively, be used for the purification of eye lens cells in accordance with the invention due to their high and relatively restricted embryonic lens expression pattern, and their relatively high expression in purified human lens cells.
5. Cell culture post cell purification
MACS purified cells were assessed for their ability to be maintained in culture. Basic fibroblast growth factor (bFGF) at low concentration has been reported to maintain LECs in culture or to stimulate lens fibre cell formation at higher concentrations (Chamberlain and McAvoy et al., 1997). The reported 3-stage lens cell differentiation method (Yang et al, 2010) employs high concentrations of bFGF in the final stage of the protocol together with the addition of Wnt-3a. As that system produced contaminating fibre cells and lentoid structures, low concentrations of bFGF was employed for maintenance of hPSC-derived LECs and to avoid stimulation of differentiation into lens fiber cells.
Cells purified for RORl expression by MACS as described above using CA1 and/or MEL1 human embryonic stem cells were cultured at differing cell densities in 1 ml of RPMI media containing 20 ng/nL bFGF in Matrigel-coated wells of a 24 well plate with 10 μΜ Ri on the day of plating. The medium was then changed the next day with RPMI containing 20 ng/mL bFGF without Ri, and changed with this medium approximately twice weekly as necessary. Cells may be cultured in DMEM, DMEM:F12, F12, Medium 199 (M199) or similar medium instead of RPMI . The cultured cells were assessed for the appearance of lentoids and micrographs taken.
Cells seeded greater than 9xl05 per well were confluent 1 day post plating although lentoids appeared after 3 days. Cells seeded at 4 xlO5 cells/well were confluent within 3 days, and lentoids did not appear until 8 days and were few. Cells seeded below 5xl04 cells per well did not appear to proliferate, lentoids appeared and a significant degree of cell death was visible.
These results show that the MACS purified RORl+ cells can be readily cultured at a concentration of 4xl05 cells/well of a 24 well plate for subsequent use as required (or approximately equivalent seeding densities of different sized culture dishes). The above observations further indicate that like primary lens epithelium, the MACS purified cells optimally require neighboring cells to proliferate and can be maintained in low bFGF conditions. Additionally, high densities of the purified cells can stimulate lentoid production. This may also mirror events in the native eye lens as throughout life the epithelium proliferates providing an ongoing supply of secondary lens fibre cells which constantly encircle the lens. In addition, these results also show that higher concentrations of bFGF may be used to stimulate LEC differentiation to fibre cells in a controlled setting.
6. Toxicology and anti-PCO assay employing MACS purified lens cells
To determine the utility of cells purified for ROR1 expression via MACS for applications such as high throughput anti-PCO drug screening and/or toxicology screening, an assay utilising hydrogen peroxide (H202) and RORl+ cells derived from CA1 human embryonic stem cells was used . All cells were sorted and plated at 4xl05 cells per well (for a total 6 wells; N=3), and allowed to proliferate for 3 days.
At confluence, 2 nM of H202 in low bFGF medium was added as treatment in 3 wells and the respective control wells had their low bFGF medium refreshed. After 3 hours cell death was visualised via light microscope in H202 treated and control wells. All wells were stained with propidium iodide (PI) and Hoechst stain, allowing dead cell nuclei and all cell nuclei respectively to be visualised. Total cell numbers and percentage cell death between control and H202 treated wells established that H202 killed all cells in much less than 3 hours of treatment and the results were statistically significant.
To further demonstrate the utility of the purified hLECs for anti-PCO drug screening, the purified hLECs were exposed to pure water in a cell death assay. Water was chosen as it has previously been used in a clinical trial to remove primary human lens epithelial cells during cataract surgery, although with no long-term reduction in the rate of PCO (Rabsiler et al. 2007. Br J Ophthalmology 91 :912-5). For this hLEC-based cell death assay, purified hLECs were seeded in 96 well-plate and 6 well-plate formats at ~4 x 104 cells/well and -1.5 x 105 cells/well (respectively). The cells were seeded in Matrigel™-coated wells in medium consisting of DMEM:F12 containing 20 ng/mL FGF2 and penicillin/streptomycin until use. To assess induction of cell death in response to exposure to water, the medium was removed and pure water added containing Hoechst 33342 and propidium iodide (each at ~1 mg/mL; -200 DL was added per well for the 96 well-plate format and ~2 mL for the 6 well-plate format). The cells were then assessed by light and fluorescent microscopy using a CKX41 microscope at the following intervals, with the number of live cells (only Hoechst stained) and dead cells (both Hoechst and propidium iodide stained) counted: 0, 2, 3 and 5 minutes, then 5 minute intervals until 60 minutes after initial exposure to water. Light and fluorescence images were taken at various time points. The results are shown in Fig. 3.
EXAMPLE 4 Evaluation of MACS purified cells
RNA-seq high-resolution gene expression profiling was used to define the genes expressed by human lens cells purified by MACs on the basis of ROR1 expression as described above using ROR1+ cells purified from differentiating CA1 human embryonic stem cell cultures. Briefly, three samples of stranded total RNA from two biological replicates of ROR1 -purified human lens cells were run on one lane of 2x100 paired-end sequencing. The resulting data was mapped and aligned to the genome using the TopHat aligner (Centre for Computational Biology, Johns Hopkins
University, Baltimore, MD 21205, USA). Two tools were then used for read quantification: i) Cufflinks, which produces FPKM values (Fragments Per Kilobase Of Exon Per Million Fragments Mapped that is, read counts normalised against gene length and the total number of reads in the library) (Trapnell C et al., 2012), and ii) HT- seq, which produces raw counts of fragments mapping to a gene (Anders S et al., 2015). Aligned sequences were then analysed against the human ENSEMBLE annotation as a reference. To assess the similarity among the three samples the Pearson correlation coefficient was established for each pair of samples. This analysis showed that all three samples had good positive correlation with each other (Pearson correlation coefficient > 0.8). To assess the lens-specificity of the samples the expression of known lens genes was specifically assessed. To perform an unbiased analysis two lens gene sets were used: (1) a manually curated lens gene set based on Table 2 in Lachke et al., 2012 (Investigative Ophthalmology and Visual Science 53(3): 1617-27); and (2) a gene set based on the most highly lens-enriched gene in embryonic mouse lens based on iSyTE (Lachke et al., 2012). In particular, the mouse embryonic lens gene set is the union of the top 100 highly ranked lens-specific genes from three mouse embryonic days (El 0.5- E12.5), and shows good correlation with the purified lens cells (Fig.4). These data clearly show that all three samples expressed large numbers of known lens genes (Fig. 5 and Fig. 6), including key lens genes such as PAX6, PROXl, CRYAA, CRYAB, MAF, FOXE3, SIX3, PITX3, etc. As expected, non-lens cell genes werenot expressed within the 3 RNA-seq including: pluripotent cell genes (e.g., NANOG, OCT4, ZIC3, ESRRB, etc.); endodermal cell genes (e.g., GATA4, GDF3, Von Willebrand factor, etc.);
mesodermal cell genes (e.g., Brachyury, Goosecoid, CD34, CXCR3, etc.); and non-lens ectodermal cell genes (e.g., RPE65, NEUROD1, Choline acetyl transferase, etc.).
To investigate whether the RNA-seq profiles are a good representative of human lens epithelial cells, the genome- wide expression profiles of the three RNA-seq profiles were compared against published microarray data from human lens epithelial cells and human lens fibre cells (Gene Expression Omnibus dataset GSE2256, Hawse JR et al., 2005). The normalised data from this dataset was log2 transformed, and the gene expression levels represented as the mean of the log2 expression of their respective biological triplicates. All three purified human lens cell RNA-seq samples were shown to have good positive correlation with the primary lens epithelial and lens fibre cell profiles, with slightly higher correlation with the epithelial cell profile. This indicates the purified human lens cells more closely resemble human lens epithelial cells than fibre cells. It was also observed that the RNAseq-microarray correlation is stronger at higher gene expression, consistent with i) the expectation that microarrays have lower resolution at the lower end of gene expression spectrum (i.e., the RNA-seq data better resolves lowly-expressed from non-expressed genes), and ii) the observation of a bimodal distribution of FPKM values for gene expression. Analysis of the expression of the 7 human lens cell purification marker proteins ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 identified in accordance with Example 2 utilising the Excel-based macro gene expression analysis and El 4.5 mouse in-situ hybridisation data (Genepaint Org, Max -Planck Institute of Biophysical Chemistry, Goettingen, Germany; www.genepaint.org) showed all are highly expressed within the purified lens cells and thus suitable for lens cell purification (see Fig. 7).
The purification of LECs in accordance with the invention provides for downstream application of the purified cells, such as the study of mechanisms of primary and secondary cataract development, the development of primary and secondary cataract drug-screening assays (which require LEC populations essentially free of contaminating non-lens cells and LFCs), and for toxicology assays. Key to the exemplified purification of LECs was the identification of cell surface proteins ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 which appear to be specifically expressed in the lens epithelium at a particular stage of embryonic development. Moreover, the use of MACS (or other cell separation technology) allows for the purification of large-scale populations of human or non-human LECs without contaminating non-lens cells in an efficient, simple, reproducible and scalable manner. The results demonstrate that these purified LECs can be maintained in culture and have the potential to be stimulated to become LFCs in a controlled manner.
Limited access to human lens cells has severely inhibited the development of needed new presbyopia and cataract treatments. The method for purification of LECs as herein described provides a long-awaited source of normal or diseased lens cells to aid the development of new accommodation-retaining, presbyopia and cataract treatments suitable for both adults and children and so has broad clinical, research and commercial applications.
Although a number of embodiments of the invention have been described above, it will be understood that various modifications and changes may be made without departing from the scope of the invention. Hence, the embodiments described above are only illustrative and not to be taken as being restrictive.. LITERATURE REFERENCES
Adewumi, O et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nature Biotechnology, 2007. Vol. 25(7):803-16).
Anders S. et al. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics, 2015, Vol. 31(2): 166-169.
Chamberlain, C. G and McAvoy, J. W. 1997. Fibre differentiation and polarity in the mammalian lens: a key role for FGF. Progress in Retinal and Eye Research. Vol. 16(3), July 1997, 443-478.
Hawse JR, DeAmicis-Tress C, Cowell TL, Kantorow M. Identification of global gene expression differences between human lens epithelial and cortical fiber cells reveals specific genes and their associated pathways important for specialized lens cell functions. Mol Vis, 2005, Apr 18; 11 :274-83.
Kolle G. et al. Identification of human embryonic stem cell surface markers by combined membrane -polysome translation state array analysis and
immunotranscriptional profiling. Stem Cells, 2009; 27:2446-2456.
Matteson, P. G., Desai, J. et al. The orphan G protein-coupled receptor, Gprl61, encodes the vacuolated lens locus and controls neurulation and lens development. Proc Natl Acad Sci U S A. 2008, 105, 6, 2088-93.
Matsuda T, Nomi M, Ikeya M, Kani S, Oishi I, Terashima T, Takada S, Minami
Y. Expression of the receptor tyrosine kinase genes, Rorl and Ror2, during mouse development. Mech Dev. 2001 Jul;105(l-2): 153-6
Lachke et al. iSyTE: Integrated systems tool for eye gene
discovery.Investigative Ophthalmology and Visual Science, March 2012, Vol.
53(3): 1617-27.
Mengarelli, I and Barberi, T. Derivation of multiple cranial tissues and isolation of lens epithelium-like cells from human embryonic stem cells. Stem Cells
Translational Medicine. 2013; 2:94-106.
O'Connor, M. D., and McAvoy, J. W. In vitro generation of functional lens-lie structures with relevance to age-related nuclear cataract. Invest. Ophthal. & Vis.
Science, Mar. 2007; Vol. 48(3): 1245-1252. Oishi I, Takeuchi S, Hashimoto R, Nagabukuro A, Ueda T, Liu ZJ, Hatta T, Akira S, Matsuda Y, Yamamura H, Otani H, Minami Y. Spatio-temporally regulated expression of receptor tyrosine kinases, mRorl, mRor2, during mouse development: implications in development and function of the nervous system. Genes Cells. 1999 Jan;4(l):41-56.
Pouton, Colin W. Haynes, John M. Embryonic stem cells as a source of models for drug discovery. Nature Reviews Drug Discovery. Aug 2007, Vol. 6 Issue 8, p605- 616.
Takahashi K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007, Vol. 131, 861-872.
Trapnell C. et al. Differential gene and transcript expression analysis of RNA- seq experiments with TopHat and Cufflinks. Nat. Protoc, 2012, 7(3):562-578.
Watanabe et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotech. 2007. 25(6):681-6.
Wormstone IM, Tamiya S, Eldred JA, Lazaridis K, Chantry A, Reddan JR,
Anderson I, Duncan G: Characterization of TGF-b2 signaling and function in a human lens cell line. Exp Eye Res 2004;78:705-714.
Wormstone IM, Tamiya S, Marcantonio JM, Reddan JR: Hepatocyte growth factor function and c-met expression in human lens epithelial cells. Inv Ophthalm Vis Scie 2000;41 :4216-4222.
Yang et al. Efficient generation of lens progenitor cells and lentoid bodies from human embryonic stem cells in chemically defined conditions. FASEB J. 2010.
24(9):3274-83.

Claims

1. A method for the purification of eye lens cells and/or progenitor cells thereof from a mixed population of cells, comprising isolating cells on the basis of outer cell membrane expression of at least one marker selected from the group consisting of RORl, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2 from the mixed cell population, and collecting the isolated cells.
2. A method according to claim 1, wherein the selected marker is RORl .
3. A method according to claim 1, wherein the selected marker is GPR161.
4. A method according to claim 1, wherein the selected marker is CD81.
5. A method according to claim 1, wherein the selected marker is ODZ3.
6. A method according to claim 1 , wherein the selected marker is SLC7A11.
7. A method according to claim 1, wherein the selected marker is SLC16A1.
8. A method according to claim 1, wherein the selected marker is SLC23A2.
9. A method according to any one of claims 1 to 8 wherein the eye lens cells and/or progenitor cells thereof express the selected marker and one or more other markers from the group consisting of RORl, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 and SLC23A2.
10. A method according to any one of claims 1 to 9, wherein the eye lens cells and/or progenitor cells thereof express at least a majority of the markers in the group.
11. A method according to any one of claims 1 to 10, wherein the eye lens cells and/or progenitor cells thereof express all of the markers in the group.
12. A method according to any one of claims 1 to 11, wherein the mixed population of cells comprises a mixed population of embryonic cells.
13. A method according to any one of claims 1 to 12, further comprising providing the mixed population of cells by inducing pluripotent stem cells to differentiate into the lens cells.
14. A method according to any one of claims 1 to 13, wherein the eye lens cells are lens epithelial cells or early differentiating lens fibre cells expressing RORl .
15. A method according to any one of claims 1 to 13, wherein the eye lens cells are lens epithelial cells or early differentiating lens fibre cells expressing GPR161.
16. A method according to any one of claims 1 to 15, wherein the isolating of the cells comprises contacting cells in the mixed cell population with a respective binding agent for binding to the selected marker, and separating the cells to which the binding agent has bound from the mixed population.
17. A method according to claim 16, wherein the binding agent comprises an antibody or a binding fragment thereof.
18. A method according to claim 16 or 17, wherein the cells bound by the binding agent are separated from the mixed population of cells by magnetic separation.
19. A method according to claim 18, wherein the magnetic separation comprises magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS).
20. A method according to any one of claims 1 to 19 wherein the cells are human cells.
21. A preparation of eye lens cells purified by a method as defined in any in any one of claims 1 to 20.
22. A purified population of eye lens cells and/or progenitor forms thereof, having outer cell membrane expression of at least one marker selected from the group consisting of ROR1, GPR161, CD81, ODZ3, SLC7A11, SCL16A1 and SLC23A2.
23. An assay for screening the effect of a test agent on eye lens cells, comprising: providing a purified preparation of eye lens cells and/or progenitor forms thereof, having outer cell membrane expression of at least one marker selected from the group consisting of ROR1, GPR161, CD81, ODZ3, SLC7A11, SCL16A1 and
SLC23A2;
treating the cells with at least one test agent;
culturing the treated cells for a predetermined period under suitable conditions for maintenance of the cells; and
assessing the effect of the test agent on the cultured cells.
24. An assay according to claim 23, wherein the assay is a toxicology assay or a drug screening assay.
25. An assay according to claim 23 or 24, wherein the eye lens cells are human or non-human cells.
26. An in-vitro eye lens prepared from a purified preparation of cells as defined in claim 21 or 22.
27. A method for providing a lentoid, comprising culturing cells as defined in claim 21 or 22 under conditions suitable for the generation of the lentoid and for a period of time to generate the lentoid.
28. A lentoid provided by a method as defined in claim 27.
PCT/AU2015/000046 2014-01-29 2015-01-29 A method for the purification of eye lens cells WO2015113110A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2014900269A AU2014900269A0 (en) 2014-01-29 A method for the purification of eye lens cells.
AU2014900269 2014-01-29

Publications (1)

Publication Number Publication Date
WO2015113110A1 true WO2015113110A1 (en) 2015-08-06

Family

ID=53756038

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2015/000046 WO2015113110A1 (en) 2014-01-29 2015-01-29 A method for the purification of eye lens cells

Country Status (1)

Country Link
WO (1) WO2015113110A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10618959B2 (en) 2016-01-20 2020-04-14 Nbe-Therapeutics Ag ROR1 antibody compositions and related methods
US10758556B2 (en) 2017-08-07 2020-09-01 Nbe-Therapeutics Ag Anthracycline-based antibody drug conjugates having high in vivo tolerability
US11845793B2 (en) 2015-10-30 2023-12-19 Nbe-Therapeutics Ag Anti-ROR1 antibodies

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BASSNETT, S. ET AL.: "The membrane proteome of the mouse lens fiber cell", MOLECULAR VISION, vol. 15, 2009, pages 2448 - 2463, XP055216180 *
CHIDLOW, G. ET AL.: "Expression of monocarboxylate transporters in rat ocular tissues", AMERICAN JOURNAL OF PHYSIOLOGY. CELL PHYSIOLOGY, vol. 288, 2005, pages C416 - C428, XP055216196 *
IVANOV, D. ET AL.: "Microarray analysis of fiber cell maturation in the lens", FEBS LETTERS, vol. 579, 2005, pages 1213 - 1219, XP029243253 *
KANEMAKI, N. ET AL.: "Establishment of a lens epithelial cell line from a canine mature cataract", EXPERIMENTAL ANIMALS / JAPANESE ASSOCIATION FOR LABORATORY ANIMAL SCIENCE, vol. 61, 2012, pages 41 - 47, XP055216205 *
MARUO, T ET AL.: "Canine amino acid transport system Xc(-): cDNA sequence, distribution and cystine transport activity in lens epithelial cells", THE JOURNAL OF VETERINARY MEDICAL SCIENCE, vol. 76, 2014, pages 523 - 530, XP055216182 *
MATTESON, P.G. ET AL.: "The orphan G protein-coupled receptor, Gpr161, encodes the vacuolated lens locus and controls neurulation and lens development", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 105, 2008, pages 2088 - 2093, XP055216178 *
MENGARELLI, I. ET AL.: "Derivation of multiple cranial tissues and isolation of lens epithelium-like cells from human embryonic stem cells", STEM CELLS TRANSLATIONAL MEDICINE, vol. 2, 2013, pages 94 - 106, XP055216164 *
O'CONNOR, M.D. ET AL.: "In vitro generation of functional lens-like structures with relevance to age-related nuclear cataract", INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, vol. 48, 2007, pages 1245 - 1252, XP055216169 *
OISHI, 1. ET AL.: "Spatio-temporally regulated expression of receptor tyrosine kinases, mRorl, mRor2, during mouse development: implications in development and function of the nervous system", GENES TO CELLS, vol. 4, 1999, pages 41 - 56, XP002306519 *
YANG, C. ET AL.: "Efficient generation of lens progenitor cells and lentoid bodies from human embryonic stem cells in chemically defined conditions", THE FASEB JOURNAL, vol. 24, 2010, pages 3274 - 3283, XP055075261 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11845793B2 (en) 2015-10-30 2023-12-19 Nbe-Therapeutics Ag Anti-ROR1 antibodies
US10618959B2 (en) 2016-01-20 2020-04-14 Nbe-Therapeutics Ag ROR1 antibody compositions and related methods
US11242388B2 (en) 2016-01-20 2022-02-08 Nbe-Therapeutics Ag ROR1 antibody compositions and related methods
US10758556B2 (en) 2017-08-07 2020-09-01 Nbe-Therapeutics Ag Anthracycline-based antibody drug conjugates having high in vivo tolerability
US12121527B2 (en) 2017-08-07 2024-10-22 Nbe-Therapeutics Ag Anthracycline-based antibody drug conjugates having high in vivo tolerability

Similar Documents

Publication Publication Date Title
Zhou et al. CD133, one of the markers of cancer stem cells in Hep‐2 cell line
Bohlen et al. Isolation and culture of microglia
Panchision et al. Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24
Dhar et al. Long-term maintenance of neuronally differentiated human adipose tissue–derived stem cells
KR102208889B1 (en) Method for controlling the differentiation of pluripotent stem cells
Choi et al. Purification of pig muscle stem cells using magnetic-activated cell sorting (MACS) based on the expression of cluster of differentiation 29 (CD29)
JP2018117582A (en) Method for evaluating quality of stem cell and kit for evaluating quality of stem cell
Hertsenberg et al. Generation of corneal keratocytes from human embryonic stem cells
US10093898B2 (en) Purification of functional human astrocytes
WO2015113110A1 (en) A method for the purification of eye lens cells
Hu et al. Characterization and retinal neuron differentiation of WERI-Rb1 cancer stem cells
Girard et al. High and low permeability of human pluripotent stem cell-derived Blood Brain barrier models depend on epithelial or endothelial features
Giannone et al. Single-cell RNA sequencing analysis of the early postnatal mouse lens epithelium
Muñiz et al. Deriving retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells by different sizes of embryoid bodies
Lenkiewicz et al. Culture and isolation of brain tumor initiating cells
Tome-Garcia et al. FACS-based isolation of neural and glioma stem cell populations from fresh human tissues utilizing EGF ligand
WO2021041858A1 (en) Differentiated tendon cells derived from pluripotent progenitor cells and methods of use thereof
EP3778871A1 (en) Method for producing stem cell-derived lacrimal gland tissue
Kumánovics et al. Flow cytometry for B-cell subset analysis in immunodeficiencies
JP2019150039A (en) Identification and isolation of human corneal endothelial cells (hcecs)
US9045735B2 (en) Enrichment of tissue-derived adult stem cells based on retained extracellular matrix material
EP3129469B1 (en) Cell-surface marker of early msc aging
Wang et al. SOX2‐positive retinal stem cells are identified in adult human pars plicata by single‐cell transcriptomic analyses
Harichandan et al. Molecular signatures of primary human spermatogonial progenitors and its neighboring peritubular stromal compartment
Jaime et al. SIX1+ PAX3+ identify a progenitor for myogenic lineage commitment from hPSCs

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15743350

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15743350

Country of ref document: EP

Kind code of ref document: A1