WO2016035874A1 - 角膜内皮細胞マーカー - Google Patents
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- WO2016035874A1 WO2016035874A1 PCT/JP2015/075164 JP2015075164W WO2016035874A1 WO 2016035874 A1 WO2016035874 A1 WO 2016035874A1 JP 2015075164 W JP2015075164 W JP 2015075164W WO 2016035874 A1 WO2016035874 A1 WO 2016035874A1
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Definitions
- the present invention relates to a molecular marker specifically expressed in corneal endothelial cells, a method for producing corneal endothelial cells using the same, a method for evaluating corneal endothelial cells, and the like.
- the cornea is a transparent tissue located on the surface of the eyeball, and in order from the surface, a corneal epithelial layer, Bowman's membrane, corneal parenchymal layer, Descemet's membrane, and corneal endothelium layer exist.
- the corneal epithelial layer is the outermost layer of the cornea and functions as a barrier that protects the cornea from dust and bacteria from the outside.
- the Bowman membrane is believed to function as the base of the corneal epithelial layer.
- the corneal stroma is the thickest of the three layers and plays a role in maintaining the strength of the cornea.
- the Descemet's membrane is a thin film below the corneal stroma and serves to connect the corneal stroma and the corneal endothelium.
- the corneal endothelium layer is a monolayer cell layer in which hexagonal corneal endothelial cells are regularly arranged in a pavement shape, and the water in the anterior chamber penetrates into the corneal stroma by the barrier function and the pump function. It plays an important role in maintaining the transparency of the cornea by keeping the water content constant by preventing water from being discharged to the anterior chamber side.
- corneal endothelial cells When corneal endothelial cells are damaged due to genetic factors or external factors, and the number of corneal endothelial cells is significantly reduced, the above functions are impaired and corneal edema is caused. Severe corneal endothelial dysfunction makes it impossible to maintain the transparency of the cornea, and when bullous keratopathy develops, it causes a marked loss of visual acuity.
- Corneal endothelial cells are extremely poor in proliferating ability in the human body. Therefore, corneal transplantation is currently the most effective means of radical treatment for severe corneal endothelial dysfunction. In fact, bullous keratopathy is the leading indication for corneal transplantation. Conventionally, full-thickness corneal transplantation has been performed for corneal endothelial dysfunction, but chronic donor shortage and rejection after transplantation are problematic. In order to alleviate rejection, a method (Descemet's Stripping Automated Endothelial Keratoplasty; DSAEK) has been developed to transplant only a part of the tissue containing the corneal endothelium into the diseased eye, but DSAEK can not overcome the problem of donor shortage . In addition, a method of proliferating corneal endothelial cells in vitro and using them for treatment has been tried (Non-patent Document 1, Patent Documents 1 to 3). It disappears (Non-Patent Document 2).
- Non-patent Document 3 cord blood-derived mesenchymal stem cells
- bone marrow-derived cells Non-patent Document 4
- iris-derived stem cells Non-patent Document 5
- corneal stroma-derived stem cells Non-patent Document 6
- corneal endothelial cells for transplantation from embryonic stem cells
- Patent Document 4 induced pluripotent stem cells
- corneal endothelial cell-specific marker for the purpose of isolating and evaluating the target product, corneal endothelial cells, is essential in these studies.
- Non-patent Document 8 proteins such as ZO-1 (Non-patent Document 8), Na + -K + -ATPase (Non-patent Document 9), and N-cadherin (Non-patent Document 10) are used as corneal endothelial cell markers. These proteins are non-specifically expressed in many other cells (Non-Patent Documents 11 to 13). Therefore, the isolation of corneal endothelial cells using these proteins is satisfactory, especially considering that the stem cells used for induction of differentiation into corneal endothelial cells have multipotency into various cells. I can't expect it.
- the present invention provides molecular markers that are specifically expressed in corneal endothelial cells, and applied techniques such as a method for producing corneal endothelial cells and a method for evaluating corneal endothelial cells using the same.
- the purpose is to do.
- the inventors of the present invention have made extensive studies to solve the above problems, and from a database that comprehensively covers genes expressed in corneal endothelial cells, the expression level in corneal endothelial cells is high, and cell membrane proteins are encoded. And those with low expression levels in other tissues were selected. Then, the present inventors confirmed the expression of these genes in corneal endothelial cells and other tissues, and found genes that are specifically expressed in corneal endothelial cells. Furthermore, the present inventors immunostained human corneal sections targeting the proteins encoded by these genes, and confirmed that they were specifically expressed on the corneal endothelium. Based on these findings, the following inventions are provided.
- Item 1 A method for producing corneal endothelial cells, comprising a step of sorting cells expressing one or more selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1 from a cell population containing corneal endothelial cells. .
- Item 2. The method according to Item 1, wherein the cell population containing corneal endothelial cells is obtained by inducing differentiation of stem cells.
- Item 3. Item 2. The method according to Item 1, wherein the cell population containing corneal endothelial cells is obtained by culturing corneal endothelial cells.
- the sorting step includes binding an antibody that specifically recognizes one or more selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1 to the cell population including the corneal endothelial cells.
- Item 4. The method according to any one of Items 1 to 3.
- Item 5. A method for evaluating corneal endothelial cells using one or more expression selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1 as an index.
- Item 6. A marker for detecting corneal endothelial cells, comprising one or more selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1.
- Item 7. A kit for detecting corneal endothelial cells, comprising a substance that specifically recognizes one or more selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CL
- corneal endothelial cells can be specifically detected. Moreover, since the expression product of the molecular marker is a cell surface protein, it can be identified and / or sorted while the cell is alive. Therefore, corneal endothelial cells can be efficiently provided in a live state. Since the corneal endothelial cells obtained in this manner are suitable for transplantation, they can not only treat a disease that requires transplantation of corneal endothelial cells, but also contribute to the elimination of donor shortages in corneal transplantation. In addition, by using the molecular marker, it is possible to appropriately evaluate whether the cultured corneal endothelial cells are in a state suitable for transplantation. Therefore, the present invention is useful for treating various diseases caused by dysfunction of corneal endothelial cells.
- CECs are corneal endothelial cells
- A-brain is adult brain
- F-brain is fetal brain
- SC is spinal cord
- SG is salivary gland
- BM is bone marrow
- SM is skeletal muscle
- A-liver is adult liver
- F- liver means fetal liver
- SI means small intestine.
- shaft shows the relative expression level compared with control.
- CECs are corneal endothelial cells
- CC are cultured corneal endothelial cells
- C. stroma is corneal parenchyma
- C. epi. Is corneal epithelium
- Limbus is corneal limbus
- Epi is iris pigment epithelial cells
- TM is Trabecular meshwork
- CB means ciliary body
- RPE / choroid means retinal pigment epithelium / choroid
- ON means optic nerve.
- shaft shows the relative expression level compared with control.
- the results of immunostaining human corneal sections for six proteins (GRIP1, CLRN1, MRGPRX3, ZP4, GLP1R, and HTR1D) are shown.
- the expression level of the ZP4 gene before and after inducing human iPS cells into corneal endothelial cells is shown.
- Cell population containing corneal endothelial cells which is a starting material for producing corneal endothelial cells, is an aggregate of cells containing corneal endothelial cells, and as long as it contains corneal endothelial cells, its origin And the configuration is not limited.
- the cell population containing corneal endothelial cells includes a cell population obtained by artificially differentiating stem cells and a cell population obtained by culturing corneal endothelial cells isolated from the cornea.
- the cell population including corneal endothelial cells often includes cells other than corneal endothelial cells, but may be composed only of corneal endothelial cells.
- the origin of the cell population containing corneal endothelial cells is not particularly limited, but is preferably human.
- the number of corneal endothelial cells contained in a cell population including corneal endothelial cells is 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% % Or more, 70% or more, or 80% or more.
- Stem cells used for preparing a cell population containing corneal endothelial cells are not particularly limited as long as they can be cultured in vitro and can be differentiated into corneal endothelial cells. It can be used by appropriately selecting from stem cells to be developed in the future. Examples of such stem cells include induced pluripotent stem cells (induced pluripotent stem cells; iPS cells), embryonic stem cells (ES cells), pluripotent stem cells derived from fetal primordial germ cells (EG cells), testis-derived Pluripotent stem cells (GS cells), human somatic stem cells (tissue stem cells) that can differentiate into corneal endothelial cells, and the like.
- induced pluripotent stem cells induced pluripotent stem cells
- ES cells embryonic stem cells
- GS cells testis-derived Pluripotent stem cells
- tissue stem cells tissue stem cells
- IPS cells can be obtained by any technique, for example, by introducing a specific reprogramming factor into a somatic cell in the form of DNA or protein.
- reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Examples include Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1.
- the reprogramming factors may be used alone or in any combination.
- somatic cell examples include any cell for which iPS cell production has been confirmed so far and any cell to be reported in the future.
- examples of preferable somatic cells in one embodiment include fibroblasts and leukocytes.
- the preferred somatic cell origin is human.
- ES cells can be obtained by any technique, for example, established by removing an inner cell mass from a blastocyst of a fertilized egg of a mammal (preferably human) and culturing the inner cell mass on a fibroblast feeder. can do.
- the mammal is not particularly limited, but is preferably a human.
- the maintenance of ES cells by subculture involves the addition of a culture solution containing substances such as leukemia inhibitory factor (LIF) and / or basic fibroblast growth factor (bFGF). Can be used.
- Selection of ES cells can be performed using, for example, expression of gene markers such as OCT-3 / 4, NANOG, ECAD and the like as an index.
- EG cells are established from embryonic primordial germ cells and have the same pluripotency as ES cells. In the presence of substances such as LIF, bFGF, and stem cell factor (stem cell factor) It can be established by culturing cells (Y. Matsui et al. (1992), Cell 70, 841-847; JL Resnick et al. (1992), Nature, 359: 550-551).
- GS cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various types of cells. It is capable of self-replication in a culture solution containing glial cell line-derived neurotrophic factor (GDNF), and by repeating subculture under the same culture conditions as ES cells, (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69: 612-616).
- GDNF glial cell line-derived neurotrophic factor
- somatic stem cells that can differentiate into corneal endothelial cells include neural crest stem cells derived from corneal stroma (COPs), mesenchymal stem cells (mesenchymal stem cells), and skin-derived pluripotent progenitor cells (skin-derived) precursors: SKPs) and the like.
- COPs and SKPs are preferred.
- COPs can be prepared, for example, by removing the epithelium and endothelium from the cornea, treating the corneal stroma with collagenase, and culturing cells separated in DMEM / F12 medium supplemented with EGF, FGF2, B27 supplement, and LIF .
- SKPs can be prepared, for example, according to the method described in NatNCell Biol., 2001 vol. 3, 778-784.
- preferred stem cells are iPS cells.
- preferred stem cells are neural crest stem cells, more preferably iPS cell-derived neural crest stem cells and corneal stroma-derived neural crest stem cells.
- Neural crest stem cells are pluripotent stem cells with self-renewal and multipotency. During the development of vertebrates, they migrate from the dorsal side of the neural tube into the body and contribute to the formation of various tissues. It is known. Induction of neural crest stem cells from iPS cells can be performed according to a technique known in the art or a method analogous thereto. For example, it can be carried out in accordance with the method described in Nature Protocols, 2010 Vol. 5, Vol. 5, No. 4, and 688-701. Differentiation into corneal endothelial cells can be efficiently induced by using neural crest stem cells.
- the method for inducing differentiation of the stem cells described above into corneal endothelial cells is not particularly limited, and can be performed using a known method and a method developed in the future.
- differentiation can be induced according to the method disclosed in Patent Document 4.
- one or more differentiation inducers selected from the group consisting of GSK3 inhibitor, retinoic acid, TGFb2, insulin and ROCK inhibitor are added to a medium suitable for stem cell culture (eg, MEM medium).
- MEM medium suitable for stem cell culture
- the cells can be differentiated into corneal endothelial cells by culturing them for several days to about one and a half months under the conditions of 30 to 40 ° C. and 1 to 10% CO 2 in the added medium.
- the differentiation inducing factor preferably includes a GSK3 inhibitor and retinoic acid, more preferably includes a GSK3 inhibitor, retinoic acid, and a ROCK inhibitor, and more preferably includes a GSK3 inhibitor, retinoic acid, a ROCK inhibitor, and insulin. including.
- a cell population containing corneal endothelial cells can be obtained from the stem cells.
- the obtained cell population containing corneal endothelial cells may be immediately used for sorting corneal endothelial cells, or may be used for sorting corneal endothelial cells after repeated subculture for a certain period.
- a means for obtaining a cell population containing corneal endothelial cells by culturing corneal endothelial cells isolated from the cornea is not particularly limited, and can be performed by appropriately selecting a known method and a method to be developed in the future. For example, it can be performed according to the method disclosed in WO2014 / 104366. Specifically, after collecting the Descemet's membrane with human corneal endothelial cells attached from the human cornea piece, chopped and minced in a medium containing about 0.2% collagenase under conditions of 5% CO 2 and 37 ° C. Incubate for ⁇ 3 hours.
- a DME medium containing 15% fetal calf serum (FCS) and 2 ng / mL basic fibroblast growth factor (bFGF) can be used. Thereafter, fibroblasts and the like are removed by centrifugal washing, and trypsin digestion is performed to obtain a cell population (primary cultured cells) containing pellet-shaped corneal endothelial cells.
- FCS fetal calf serum
- bFGF basic fibroblast growth factor
- the cell population containing corneal endothelial cells obtained as described above can be further cultured in a basal medium such as D-MEM or MEM generally used for culturing animal cells.
- the concentration of glucose contained in the medium is preferably 2.0 g / L or less, more preferably 0.1 to 1.0 g / L. It is preferable to add hepatocyte growth factor (HGF), epidermal growth factor (EGF), recombinant EGF (rEGF), and / or fibroblast growth factor (FGF), etc. as growth factors to the medium. These factors may be used alone or in combination as appropriate.
- the concentration of the growth factor in the medium is usually 1 to 100 ng / mL, preferably 2 to 5 ng / mL. From the viewpoint of efficiently culturing corneal endothelial cells, it is preferable to add 5 to 1,000 ⁇ g / mL of an ascorbic acid derivative such as ascorbic acid 2-phosphate in the medium.
- Corneal endothelial cells are cultured in a culture vessel coated with a matrix such as Matrigel or collagen (eg, dish, petri dish, tissue culture dish, multi-dish, microplate, microwell plate, multiplate, multiwell plate, chamber slide). , Petri dishes, tubes, trays, culture bags, etc.).
- the culture temperature is usually 35 to 38 ° C, preferably 37 ° C.
- the wet conditions are usually 90 to 100% wet, preferably 100% wet.
- the CO 2 concentration is usually 5 to 15%, preferably 10%. Therefore, it is preferable to culture in an incubator that can maintain such conditions.
- the culture period is not particularly limited. For example, the culture period can be performed until the cells become confluent (steady state) (for example, 1 to 5 days).
- the cells When the cells reach confluence, they can be subcultured as necessary. For example, confluent cells are washed with PBS, dispersed in trypsin / EDTA, centrifuged, and seeded in a culture vessel containing the same medium as described above to a cell density of 500-60,000 cells / cm 2. And can be cultured under the above-mentioned culture conditions. Further, when the cells reach confluence, the same passage operation can be repeated. In this way, a cell population containing corneal endothelial cells that are subcultured cells can be obtained.
- corneal endothelial cells Sorting of corneal endothelial cells using molecular markers as an index ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1 and PCDHB7 are specifically expressed in corneal endothelial cells Yes. Therefore, corneal endothelial cells can be produced by sorting corneal endothelial cells from a cell population containing corneal endothelial cells using at least one expression selected from these molecules as an index.
- ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1, and PCDHB7 are also referred to as molecular markers.
- ZP4 is a gene that encodes one of the glycoproteins that make up the zona pellucida, the extracellular matrix that surrounds the oocyte (Hartmann JF, et al., (1972), Proc Natl Acad Sci USA, 69: 2767) -2769). Most of them are extracellular, but the carboxyl terminus is the transmembrane domain (Gupta SK, et al. (2012), Cell Tissue Res, 349: 665-678).
- MRGPRX3 is a gene that encodes one of the Mas-related G protein-coupled receptors and has been suggested to be involved in pain control in sensory nerves (Lembo PM, et al. (2002), Nat Neurosci 5: 201-209).
- GRIP1 is a gene that encodes a protein that binds to the intracellular domain carboxyl terminus of AMPA ( ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionate) type glutamate receptor, and is expressed in synapses and the brain. (Dong H, et al., (1999), J Neurosci, 19: 6930-6941).
- AMPA ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionate
- GLP1R is a gene encoding a receptor for Glucagon-like peptide-1 which is one of the incretins that promote insulin secretion from pancreatic ⁇ cells (Orskov ⁇ C, (1992)). ), Diabetologia, 35: 701-711). Therefore, GLP1R has been reported to be expressed in the pancreas (Tornehave D, et al., (2008), J Histochem Cytochem, 56: 841-851).
- HTR1D is a gene that encodes one of the serotonin receptors and is expressed in nerve fibers of craniofacial tissue, and has been suggested to be involved in the development of migraine (Harriott AM, et al., ( 2008), Cephalalgia, 28: 933-944).
- CLRN1 has been reported as one of the causative genes of Usher syndrome typeIIIa that causes inner ear disorders and retinitis pigmentosa (Kremer H, et al., (2006), Hum Mol Genet 15 Spec No 2: p. 262- 270). CLRN1 is expressed in inner ear hair cells and retinal glial cells, and is thought to play an important role in the development and differentiation of inner ear hair cells (Geller SF, et al., (2009) , PLoS Genet 5: e1000607), but its detailed functions are not disclosed.
- SCNN1D is a gene that encodes the ⁇ subunit of epithelial Na channel (ENaC). It has been reported to be expressed in the brain, heart, respiratory organ, kidney, etc. (Ji HL et al., (2012), Am J Physiol Lung Cell Mol Physiol. 303: 1013-1026).
- PKD1 is a gene encoding polycystin-1 and has been reported as one of the causative genes of autosomal dominant polycystic kidney disease (Gabow PA, (1993) N Engl J Med. 329: 332-342). Therefore, it is highly expressed in the kidney. Polycystin-1 has been suggested to be involved in the differentiation of tubular epithelial cells (Boletta A et al., (2003) Trends Cell Biol. 13: 484-492).
- CNTN6 is a gene encoding contactin-6.
- Contactin 6 is a member of the immunoglobulin superfamily and is involved in cell adhesion and has been implicated in the formation of synaptic connections at the developmental stage of the nervous system (http: //www.ncbi.nlm.nih .gov / gene / 27255).
- NSF is a gene that encodes an N-ethylmaleimide-sensitive factor that forms a complex with SNAP (soluble NSF attachment protein) and SNARE (SNAP receptor) to promote fusion of synaptic vesicles and cell membranes (Sollner T et al., (1993) Nature. 362: 318-324).
- CNTN3 is a gene that encodes contactin 3, but its structure suggests that it is involved in cell adhesion like contactin 6. Expression in frontal lobe, occipital lobe, cerebellum, etc. has been reported (http://www.uniprot.org/uniprot/Q9P232).
- PPIP5K1 is a gene encoding inositol kinase, which has been suggested to play an important role in intracellular signal transduction (Gokhale NA et al., (2013) Biochem J. 453: 413-426).
- PCDHB7 is a gene encoding protocadherin ⁇ 7. Although the specific function is unknown, it has been suggested to play an important role in cell-cell adhesion in the nervous system (http://www.ncbi.nlm.nih.gov/gene/56129).
- the molecular marker used as an indicator of corneal endothelial cells may be only one selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1 and PCDHB7 Two or more types may be used in any combination.
- preferred molecular markers are ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1 from the viewpoint that the expression product is a surface protein of corneal endothelial cells and the specificity of expression in corneal endothelial cells is high.
- ZP4 More preferably ZP4, MRGPRX3, GRIP1, GLP1R and HTR1D, more preferably ZP4, MRGPRX3, GRIP1, and GLP1R, still more preferably ZP4, MRGPRX3, and GRIP1, more preferably ZP4 and MRGPRX3, particularly preferably ZP4.
- SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1 and PCDHB7 are thought to be expressed in other tissues such as brain, spinal cord, skeletal muscle, or liver as shown in FIG. Controls the type of stem cells from which the cell group is derived and / or conditions for differentiation into corneal endothelial cells, or adopts a cell group obtained by culturing corneal endothelial cells as a cell group containing corneal endothelial cells By doing so, the expression in those tissues can be ignored. Therefore, these molecular markers are also useful for sorting or detecting corneal endothelial cells.
- SCNN1D, PKD1, CNTN6, NSF, CNTN3, and PPIP5K1 more preferably SCNN1D, PKD1, CNTN6, NSF, and CNTN3, and more preferably SCNN1D, PKD1, CNTN6, and NSF.
- SCNN1D, PKD1, and CNTN6 are more preferable, SCNN1D and PKD1 are more preferable, and SCNN1D is particularly preferable.
- Including corneal endothelial cells using as an index the expression of one or more molecular markers selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1 and PCDHB7 Sorting of corneal endothelial cells from a cell population, for example, if the cell population does not contain cells that express the at least one molecular marker other than corneal endothelial cells, express the at least one molecular marker. This can be done by sorting the existing cells.
- the at least one molecular marker other than corneal endothelial cells when other cells that express the at least one molecular marker other than corneal endothelial cells are present in the cell population, by sorting out cells that exhibit significantly higher expression than the other cells. A corneal endothelial cell can be obtained.
- the molecular marker used as an index can be used in an appropriate combination of two or more, three or more, four or more, or five or more.
- Detecting the expression of molecular markers can be performed using any means.
- a typical detection method for example, a method of measuring the expression of a gene encoding one or more molecular markers using RT-PCR, and the presence of the one or more molecular markers are specifically bound thereto.
- the method include detection using a substance. All of the above molecular markers are surface proteins of corneal endothelial cells. Therefore, from the viewpoint that the molecular marker can be detected while the cells are alive, a detection method using a substance that specifically binds to the molecular marker is preferable.
- the type of the substance that specifically binds to the molecular marker is not particularly limited, and examples thereof include antibodies and aptamers.
- a preferable substance is an antibody or a fragment thereof.
- the antibody may be a polyclonal or monoclonal antibody. Examples of antibody fragments include Fab fragments, F (ab) 2 fragments, ScFv fragments and the like.
- the substance that specifically binds to a molecular marker preferably has a property that is not degraded by a treatment (for example, treatment with a protease) for separating a cell group including corneal endothelial cells into individual cells. .
- a treatment for example, treatment with a protease
- the antibody that specifically recognizes the molecular marker may be a commercially available one, or may be prepared using a known technique.
- the production of antibodies is well known.
- a polyclonal antibody a non-human animal is immunized with each purified molecular marker or a partial peptide thereof, and can be obtained from the serum of the animal according to a conventional method.
- a monoclonal antibody it can be obtained from a hybridoma prepared by cell fusion of spleen cells and myeloma cells obtained from an immunized animal.
- the substance is preferably labeled with an arbitrary labeling substance.
- the labeling substance is not particularly limited, and examples thereof include a fluorescent label, a radioactive label, a chemiluminescent label, an enzyme, biotin, and streptavidin.
- the substance that specifically binds to the molecular marker may be indirectly labeled.
- a pre-labeled antibody (secondary antibody) that specifically binds to the substance can be used.
- the method for identifying and sorting cells bound with a substance that specifically binds to a molecular marker is not particularly limited, and can be appropriately selected from known methods and methods developed in the future.
- the cell identification / sorting method can be selected according to the type of labeling substance used.
- Typical cell identification / sorting methods include fluorescence-activated cell sorting (Fluorescence-Activated Cell Sorting: FACS), magnetic cell sorting (Magnetic-Activated Cell Sorting: MACS), and affinity chromatography. It is.
- a fluorescence-labeled cell separation method and a magnetic cell separation method are preferred, and FACS using a flow cytometer with a cell sorter is more preferred.
- the mode of FACS is not particularly limited.
- the mode may be either a droplet charging system or a cell capture system. In this way, corneal endothelial cells can be purified.
- corneal endothelial cells can be obtained by separating corneal endothelial cells from a cell population containing corneal endothelial cells using a molecular marker as an index.
- the corneal endothelial cells also include corneal endothelial progenitor cells. According to the above method, it is possible to obtain a cell population enriched in corneal endothelial cells.
- the percentage of corneal endothelial cells contained in the cell population enriched with corneal endothelial cells is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more in terms of the number of cells. 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more.
- Corneal Endothelial cells obtained using the molecular marker as an index can be used for the treatment of diseases caused by dysfunction of the corneal endothelial layer. Therefore, the pharmaceutical composition for the treatment of corneal endothelial disease containing the corneal endothelial cell can be provided.
- Such a pharmaceutical composition may further contain various components for assisting in the maintenance, proliferation, and administration to the affected area of corneal endothelial cells, scaffold materials, carriers and the like.
- components that assist cell maintenance and / or proliferation include medium components such as carbon sources, nitrogen sources, vitamins, minerals, salts, and various cytokines.
- the scaffold material that assists administration to the affected area include collagen, polylactic acid, hyaluronic acid, cellulose, and derivatives thereof. Two or more of these components and scaffold materials can be combined.
- the pharmaceutical composition may be in the form of containing corneal endothelial cells in an aqueous solution for injection (eg, physiological saline, physiological buffer such as PBS, isotonic solution containing glucose and other adjuvants).
- a corneal endothelial cell sheet can be provided by culturing corneal endothelial cells obtained using the molecular marker as an indicator on an appropriate carrier (for example, a polymer membrane).
- a carrier for example, collagen, atelocollagen, alkali-treated collagen, gelatin, keratin, hyaluronic acid, glycosaminoglycan (chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, heparin, keratan sulfate) , Proteoglycans, alginic acid, chitosan, polyamino acids (polylactic acid), biopolymers such as cellulose, (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, vinyl ether derivatives, or these A temperature-responsive polymer such as
- Corneal endothelial cells can be cryopreserved by adding a protective agent or the like, if necessary.
- the protective agent include glycerol, DMSO (dimethyl sulfoxide), propylene glycol, acetamide and the like.
- corneal endothelial cells can be subjected to heat treatment and / or radiation treatment.
- a corneal endothelial cell, a pharmaceutical composition containing the corneal endothelial cell, and a disease that can be treated using the corneal endothelial cell sheet are not particularly limited, but include, for example, corneal endothelial dysfunction including bullous keratopathy, corneal dystrophy, development Glaucoma, Rieger malformation, congenital hereditary corneal endothelial dystrophy, limbal delmoid, keratoplasty cornea, corneal abnormalities such as keratoconus and perousoid corneal degeneration, corneal scar, corneal infiltration, corneal deposition, corneal edema, corneal ulcer, chemical Examples include eye diseases such as eye trauma due to substances and heat, keratitis, corneal degeneration, corneal infections, neuroblastoma, Hirschsprung disease, Waadenburg syndrome, and Recklinghausen disease.
- corneal endothelial dysfunction including bullous keratopathy, corneal dystrophy, development Glaucoma, Rieger
- the above-mentioned disease can be treated by transplanting a corneal endothelial cell or a corneal endothelial cell sheet into a patient who needs to transplant a corneal endothelial cell (for example, a patient suffering from the above-mentioned disease). Therefore, the treatment method of the said disease including administering the corneal endothelial cell obtained by the above-mentioned method to the patient who needs the transplant of a corneal endothelial cell is provided.
- Method for evaluating corneal endothelial cells One or more selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1 and PCDHB7 described above are specific to corneal endothelial cells Is expressed. Therefore, a method for evaluating corneal endothelial cells using the expression of the molecular marker as an index is provided.
- the expression of the molecular marker is measured and a specific expression is confirmed (or a significant expression is confirmed) for a test cell that is considered (or presumed) to be a corneal endothelial cell ).
- the test cell can be evaluated as a corneal endothelial cell.
- Preferred molecular markers for use in such an evaluation method are the same as the molecular markers described in 1-2 above.
- the test cell is not particularly limited, but for example, the above 1-1. And cells obtained by culturing the cells described in 1) on a medium and conditions suitable for inducing differentiation into corneal endothelial cells, and cells obtained by culturing corneal endothelial cells collected from the cornea.
- the method for measuring the expression of the molecular marker is not particularly limited, and for example, the above 1-2. And the measurement method using RT-PCR and the method of measuring using a substance that specifically binds to a molecular marker. By evaluating the test cell in this way, it can be safely used as a corneal endothelial cell.
- Corneal endothelial cell detection kit Corneal endothelial cells containing a substance that specifically recognizes one or more molecular markers selected from the group consisting of ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, CLRN1, SCNN1D, PKD1, CNTN6, NSF, CNTN3, PPIP5K1 and PCDHB7 A detection kit is provided.
- the substance that specifically recognizes the molecular marker is not particularly limited, and for example, 1-2. And the aptamer described in the above.
- the kit can contain any substance, container, instructions, and the like that are used for detecting corneal endothelial cells.
- the kit can include a labeling substance that labels a substance that specifically recognizes the above-described molecular marker.
- KRT3 and KRT12 which are corneal epithelial cell-specific markers, and it was confirmed that the expression level was high in data from a 56-year-old adult donor. Therefore, since it was considered that the sample derived from this individual was mixed with corneal epithelial cells, the following analysis was performed using only data derived from the remaining four individuals.
- RNA-seq reads were aligned to the human reference genome (hg19) using TopHat (version 1.4.1) and the results were used to construct a transcript model with the Cufflinks package (version 2.1.1) (Trapnell Et al., (2012), Nat. Protoc.c7: 562-578).
- Cufflinks the gene expression level was quantified as FPKM (Fragments Per Kilobase of exon per Million mapped fragments: FPKM), and genes expressing 10 FPKM or more in corneal endothelial cells were selected. As a result, 10,627 genes were selected. Next, these genes were narrowed down to only genes encoding cell membrane proteins using the GO terms shown in Table 1 below.
- RNA expression levels of candidate molecules in ocular and systemic tissues To confirm the expression levels of the 13 genes selected in 1 above in corneal endothelial cells and systemic tissues, RNA of each gene in adult human corneal endothelial cells and 22 tissues The expression level was measured by quantitative PCR. A specific procedure will be described later. As a result, as shown in FIG. 1, it was confirmed that all genes were expressed in corneal endothelial cells in accordance with the data analysis results. Among them, ZP4, MRGPRX3, GRIP1, GLP1R, HTR1D, and CLRN1 have the highest expression levels in corneal endothelial cells compared to the expression levels in other tissues, and are confirmed to be expressed only in other limited tissues. It was done.
- the expression level between these eye tissues was measured by quantitative PCR for these six genes.
- eye tissue four eyeballs obtained from two adult donors were used.
- high expression in corneal endothelial cells was confirmed for all genes.
- the corneal stroma is adjacent to the corneal endothelial layer and is derived from the cranial nerve crest like corneal endothelial cells, it is important as a marker for corneal endothelial cells that it is not expressed or is low in expression level. This is considered to be one of the features.
- CLRN1 was slightly expressed in the corneal stroma, but expression of the other five genes was not observed. Therefore, all these molecules are useful as markers specific for corneal endothelial cells.
- RNA extraction from corneal endothelial cells was performed by the following procedure. All human samples were handled in compliance with the Declaration of Helsinki. Laboratory cornea pieces and whole eyeballs from human donors were obtained from Sight Life (Seattle, WA). The cornea pieces were immersed in Optisol-GS (Bausch® & Lomb, Rochester, NY), stored at 4 ° C., and used within 4 days after death. The donor's age was 58 years. After the cornea piece was washed three times with phosphate buffered saline (PBS), the corneal endothelium and the Desme membrane were peeled off along the Schwalbe line using an insulator. RNA was extracted from the detached corneal endothelium using QiagenQmiRNeasy Mini Kit (QIAGEN Inc.).
- PBS phosphate buffered saline
- RNA extraction from cultured corneal endothelial cells was performed by the following procedure.
- a cornea piece was obtained from 4 eyes derived from 4 donors. Donor ages ranged from 14 to 25 years.
- Corneal endothelium and Descemet's membrane were isolated as described above. The isolated tissue was incubated at 37 ° C. in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 1.2 U / mL dispase II (Joint Shusei Co., Ltd.) and 1% Antibiotic-Antimycotic (Anti-Anti; Invitrogen / Gibco).
- DMEM Dulbecco's modified Eagle's medium
- Anti-Anti Anti-Anti
- Invitrogen 1% Antibiotic-Antimycotic
- Isolated corneal endothelial cells are collected by gentle centrifugation, 50 U / mL penicillin, 50 ⁇ g / mL streptomycin, 10% fetal calf serum (ICN Biomedicals, Inc., Aurora, Ohio) and 2 ng / mL basic fibroblasts After suspending in DMEM medium supplemented with cell growth factor (bFGF; invitrogen), seeded on a culture dish coated with cell adhesion reagent (FNC coating mix; Athena ES, Baltimore, MD) and 5% CO The cells were cultured at 37 ° C. in a humidified atmosphere of 2 . Thereafter, RNA was extracted using an Isogen RNA extraction kit. The cells used for RNA extraction were all from the first passage.
- bFGF cell growth factor
- FNC coating mix Athena ES, Baltimore, MD
- RNA extraction from human eye tissue was performed according to the following procedure. Four eyeballs from two donors were stored at 4 ° C. in wet containers and used within 5 days after death. Donor ages were 75 and 79 years. First, a cornea piece was prepared, and the ciliary body, iris and lens were isolated from the anterior segment, and the iris parenchyma and iris pigment epithelial cells were isolated. The corneal endothelium and Descemet's membrane were detached by the method described above, and the trabecular meshwork was isolated. Furthermore, the conjunctiva was excised from the cornea piece, and the central and limbal parts separated using 8.0 mm diameter trepan were treated overnight at 4 ° C.
- the limbal epithelium was isolated from the parenchyma.
- the nerve retina was detached from the posterior eye using an insulator, and then the retinal pigment epithelial cells (RPE) and the choroid were excised in one lump, and finally the optic nerve was isolated. All of these isolated tissues were extracted using the ISOGEN RNA extraction kit.
- human total RNA master panel II (# 636643; Clontech, Mountain View, Calif.) was obtained. Since this did not include kidney RNA and pancreatic RNA, Human Kidney Total RNA (# AM7976; Ambion, Austin, TX) and Human Pancreas Total RNA (# AM7954; Ambion) were purchased separately.
- RT-qPCR Reverse transcription quantitative polymerase chain reaction
- corneal endothelial cells Staining of corneal endothelial cells in corneal tissue
- corneal tissue fragments derived from human donors were immunostained using the antibodies shown in Table 4 below.
- the corneal endothelial layer was strongly stained when any protein was stained as a target.
- anti-ZP4 antibody, anti-GLP antibody, and anti-HTR1D antibody only the corneal endothelial layer was specifically stained.
- the corneal stroma was also stained, but the fluorescence intensity was clearly lower compared to that of corneal endothelial cells, so based on this significant difference in expression level, corneal endothelial cells Can be distinguished from the corneal stroma.
- the above six genes and the proteins encoded by them are useful as markers that specifically recognize corneal endothelial cells.
- the above immunostaining was performed according to the following procedure.
- the cornea piece was used within 11 days after death. The donor's age was 27 years.
- the cornea piece was embedded in an OCT compound, and the frozen section was cut to 10 ⁇ m using a microtome-cryostat (HM560, Thermo Fisher Fisher Inc., Waldorf, Germany). After drying at room temperature for 30 minutes, the tissue section was washed 3 times with Tris buffered saline (TBS; Takara Bio Inc.) and 1 hour with TBS containing 5% donkey serum and 0.3% Triton X-100. Incubation inhibited non-specific reactions. Each section was then incubated overnight at 4 ° C. with the primary antibodies listed in Table 4.
- the sections were further washed 3 times with TBS, together with a 200-fold dilution of each Alexa Fluor 488-conjugated secondary antibody (Life Technologies) and a 100-fold dilution of Hoechst 33342 (# B2261, Sigma-Aldrich) at room temperature. Incubated for 2 hours. The sections were observed using a fluorescence microscope (Axio Observer D1; Carl Zeiss Jena Gmbh, Jena, Germany).
- ZP4 is a useful marker for production of corneal endothelial cells and evaluation of corneal endothelial cells.
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Abstract
Description
角膜内皮細胞を含む細胞集団から、ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される一種以上を発現している細胞を分取する工程を含む、角膜内皮細胞の製造方法。
項2.
角膜内皮細胞を含む細胞集団が幹細胞を分化誘導して得られる、項1に記載の方法。
項3.
角膜内皮細胞を含む細胞集団が角膜内皮細胞を培養して得られる、項1に記載の方法。項4.
分取する工程が、ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される1種以上を特異的に認識する抗体を該角膜内皮細胞を含む細胞集団に結合させることを含む、項1~3のいずれかに記載の方法。
項5.
ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される一種以上の発現を指標にして角膜内皮細胞を評価する方法。
項6.
ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される一種以上からなる角膜内皮細胞検出用マーカー。
項7.
ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される1種以上を特異的に認識する物質を含む、角膜内皮細胞検出用キット。
1-1.角膜内皮細胞を含む細胞集団
角膜内皮細胞を製造する出発材料となる「角膜内皮細胞を含む細胞集団」とは、角膜内皮細胞を含む細胞の集合体であり、角膜内皮細胞を含む限り、その由来及び構成は制限されない。例えば、角膜内皮細胞を含む細胞集団には、幹細胞を人為的に分化させて得られた細胞集団、及び、角膜から単離された角膜内皮細胞を培養して得られた細胞集団が含まれる。角膜内皮細胞を含む細胞集団は、多くの場合、角膜内皮細胞以外の細胞を含むが、角膜内皮細胞のみで構成されていても良い。角膜内皮細胞を含む細胞集団の由来は、特に制限されないが、好ましくはヒトである。
角膜内皮細胞には、ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、CLRN1、SCNN1D、PKD1、CNTN6、NSF、CNTN3、PPIP5K1及びPCDHB7が特異的に発現している。よって、これらの分子から選択される少なくとも一種の発現を指標にして角膜内皮細胞を含む細胞集団から角膜内皮細胞を分取し、角膜内皮細胞を製造することが出来る。尚、本書では、ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、CLRN1、SCNN1D、PKD1、CNTN6、NSF、CNTN3、PPIP5K1及びPCDHB7を分子マーカーとも称する。
上記分子マーカーを指標にして得られる角膜内皮細胞は、角膜内皮層の機能障害に起因する疾患の治療に用いることができる。よって、当該角膜内皮細胞を含む、角膜内皮疾患の治療用医薬組成物を提供することができる。このような医薬組成物には、角膜内皮細胞の維持、増殖、患部への投与を補助するための各種成分、足場材料、及び担体等を更に含めてもよい。
上述するZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、CLRN1、SCNN1D、PKD1、CNTN6、NSF、CNTN3、PPIP5K1及びPCDHB7から成る群より選択される1種以上は、角膜内皮細胞に特異的に発現している。よって、当該分子マーカーの発現を指標にした角膜内皮細胞の評価方法が提供される。例えば、角膜内皮細胞であると考えられる(又は推測される)被検細胞について、当該分子マーカーの発現を測定し、特異的な発現が確認された場合(又は、有意な発現が確認された場合)に、被検細胞を角膜内皮細胞であると評価することができる。このような評価方法での使用に好ましい分子マーカーは、上記1-2で記載した分子マーカーと同じである。
ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、CLRN1、SCNN1D、PKD1、CNTN6、NSF、CNTN3、PPIP5K1及びPCDHB7から成る群より選択される1種以上の分子マーカーを特異的に認識する物質を含む角膜内皮細胞検出用キットが提供される。分子マーカーを特異的に認識する物質とは、特に制限されず、例えば、上記1-2.に記載する抗体、及びアプタマー等を挙げることができる。キットには、分子マーカーを特異的に認識する物質以外に、角膜内皮細胞の検出に利用される任意の物質、容器、及び説明書などを含めることができる。例えば、キットには、上述する分子マーカーを特異的に認識する物質を標識する標識物質を含めることができる。
1.候補分子の同定
ヒト生体内の角膜内皮細胞に発現している遺伝子の情報として、Chen et al.によって報告されたRNA-seqデータ(GSE41616, Chen, et al.,(2013), Hum Mol Genet 22: 1271-1279)を遺伝子発現情報データベース(Gene Expression Omnibus:GEO)から入手した。このデータには、3個体の成人ドナー(31歳、56歳、64歳)由来の角膜内皮細胞、並びに、2個体の胎児ドナー(16~18週)由来の角膜内皮細胞で発現しているRNAのデータが含まれている。データの信頼性を確認するため、角膜上皮細胞特異的マーカーであるKRT3及びKRT12について当該データを調べたところ、56歳の成人ドナー由来のデータでこれらの発現レベルが高いことが確認された。よって、この個体由来のsampleには、角膜上皮細胞が混在していたものと考えられたため、残りの4個体に由来するデータのみを用いて以下の解析を行った。
上記1で絞り込んだ13の遺伝子の角膜内皮細胞及び全身組織での発現量を確認するため、成人ヒト角膜内皮細胞及び22の組織における各遺伝子のRNA発現量を定量PCRにより計測した。具体的な手順は後述する。その結果、図1に示すように、全ての遺伝子が、データ解析結果に則し、角膜内皮細胞で発現していることが確認された。中でもZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、CLRN1、は、角膜内皮細胞における発現量が他の組織における発現量と比較して最も高く、他の限られた組織でしか発現していないことが確認された。
上記6つの遺伝子について、タンパク質レベルでの発現を確認するために、ヒトドナー由来の角膜組織片を下記の表4に示す抗体を用いて免疫染色を行った。その結果、図3に示す通り、いずれのタンパク質を標的として染色した場合も角膜内皮層が強く染色された。特に、抗ZP4抗体、抗GLP抗体、及び抗HTR1D抗体の3種については、角膜内皮層のみが特異的に染色された。GRIP1、CLRN1、及びMRGPRX3については、角膜実質も染色されたが、その蛍光強度は、角膜内皮細胞のものと比較して明らかに低いため、この有意な発現レベルの差に基づいて、角膜内皮細胞を角膜実質と区別することは可能である。このように、上記6つの遺伝子及びそれがコードするタンパク質が角膜内皮細胞を特異的に認識するマーカーとして有用であることが示された。
多能性幹細胞であるヒトiPS細胞(京都大学iPS細胞研究所から提供)から角膜内皮細胞を誘導したところ、図4に示す通り、誘導によってZP4の発現が上昇することが確認された。ZP4遺伝子の発現は、RT-PCRで測定した。角膜内皮細胞及び前駆細胞に特異的にZP4が発現していることから、この結果は、ZP4が角膜内皮細胞の製造及び角膜内皮細胞の評価に有用なマーカーであることを裏付けている。
Claims (7)
- 角膜内皮細胞を含む細胞集団から、ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される一種以上を発現している細胞を分取する工程を含む、角膜内皮細胞の製造方法。
- 角膜内皮細胞を含む細胞集団が幹細胞を分化誘導して得られる、請求項1に記載の方法。
- 角膜内皮細胞を含む細胞集団が角膜内皮細胞を培養して得られる、請求項1に記載の方法。
- 分取する工程が、ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される1種以上を特異的に認識する抗体を該角膜内皮細胞を含む細胞集団に結合させることを含む、請求項1~3のいずれかに記載の方法。
- ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される一種以上の発現を指標にして角膜内皮細胞を評価する方法。
- ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される一種以上からなる角膜内皮細胞検出用マーカー。
- ZP4、MRGPRX3、GRIP1、GLP1R、HTR1D、及びCLRN1から成る群より選択される1種以上を特異的に認識する物質を含む、角膜内皮細胞検出用キット。
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