WO2015113110A1 - Procédé de purification de cellules de cristallin - Google Patents

Procédé de purification de cellules de cristallin Download PDF

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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
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cells
lens
cell
eye lens
purified
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PCT/AU2015/000046
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English (en)
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Michael O'connor
Patricia Murphy
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University Of Western Sydney
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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/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye 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.

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Abstract

L'invention concerne un procédé de purification de cellules de cristallin et/ou de cellules progénitrices de ces dernières à partir d'une population mixte de cellules, comprenant l'isolement des cellules sur la base de l'expression par la membrane cellulaire externe d'au moins un marqueur choisi dans le groupe constitué par ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 et SLC23A2 à partir de la population mixte de cellules, et le recueil des cellules isolées. L'invention concerne également des préparations purifiées de cellules exprimant un ou plusieurs des marqueurs de la surface cellulaire ROR1, GPR161, CD81, ODZ3, SLC7A11, SLC16A1 et SLC23A2, et de la descendance des cellules purifiées, ainsi que des structures lentoïdes et de type cristallin préparées à partir des cellules purifiées ou de la descendance de ces dernières et des procédés pour la fourniture des structures lentoïdes et de type cristallin.
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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
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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

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