WO2013012087A1 - 角膜内皮細胞の調製方法 - Google Patents
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Definitions
- the present invention relates to a method for selectively proliferating corneal endothelial progenitor cells from a group of cells isolated from corneal endothelial tissue, and a method for obtaining corneal endothelial cells from corneal endothelial progenitor cells grown by the above method.
- the cornea is composed of five layers from the outside: a corneal epithelial layer, a Bowman membrane, a parenchymal corneal layer, a Descemet's membrane, and a corneal endothelium layer.
- the innermost corneal endothelium layer is a single cell layer, taking in substances necessary for the cornea from the anterior aqueous humor, draining the corneal water into the anterior aqueous humor, keeping the corneal thickness constant, Maintains corneal transparency.
- the number of corneal endothelial cells decreases, water drainage becomes insufficient, which causes corneal opacity and corneal endothelial diseases such as bullous keratopathy.
- the density of human corneal endothelial cells is about 3000 cells / mm 2 , but in corneal endothelial diseases, the density decreases to about 500 cells / mm 2 .
- human corneal endothelial cells do not proliferate in vivo, once they are damaged or markedly reduced, there is no method other than transplantation for the fundamental treatment.
- Non-patent Document 1 Patent Documents 1 and 2, etc.
- Non-patent Document 2 Attempts have also been made to grow corneal endothelial cells in vitro and use them for treatment.
- Non-patent Document 2 Attempts have also been made to grow corneal endothelial cells in vitro and use them for treatment.
- Non-patent Document 2 Attempts have also been made to grow corneal endothelial cells in vitro and use them for treatment.
- Non-patent Document 2 Non-patent Document 2
- corneal endothelial progenitor cells cannot be selectively proliferated with existing methods, and only differentiated cells are only temporarily proliferating.
- Non-patent Document 3 and Patent Document 3 the aggregate obtained by this method does not express the neural crest stem cell marker p75, which is the origin of the corneal endothelium, and its undifferentiation is unknown.
- p75 is expressed only in a small number of stem cells existing in adults including neural crest stem cells, and is one of the most reliable markers as an undifferentiated index.
- the subject of the present invention is to selectively proliferate undifferentiated corneal endothelial progenitor cells from a group of cells derived from corneal tissue, and to apply the corneal endothelial cells obtained by inducing it to the treatment of corneal endothelial disease,
- the purpose is to solve the problems of donor shortage and rejection in corneal transplantation.
- the inventors can selectively proliferate corneal endothelial progenitor cells by culturing cells isolated from corneal endothelial tissue by enzyme treatment at a low density for a long time using a specific serum-free medium. I found out.
- the present invention relates to a method for preparing corneal endothelial progenitor cells, characterized in that a cell group isolated from corneal endothelial cell tissue is adhered and cultured using a serum-free medium at a low density.
- the cell has a density of at least less than 10,000 cells / cm 2 , preferably 5000 cells / cm 2 or less, more preferably 20 to 2000 cells / cm 2 , still more preferably 50 to 1000 cells / cm 2 , most preferably about 100 to 500 cells / cm 2. Inoculate and culture.
- a group of undifferentiated corneal endothelial progenitor cells having high proliferative ability is selectively proliferated.
- the proliferated corneal endothelial progenitor cell group is characterized by positive neural crest marker p75, which is an undifferentiated index, and further, expression of neural crest markers FOXC2, SOX9 and corneal endothelial marker N-cadherin is also observed. It is done. These markers are highly expressed at the beginning of the culture, but disappear gradually as the growth proceeds.
- the proliferated cell group becomes highly expressed with the proliferation marker Ki-67 that is not expressed at the initial stage of culture, has a very high proliferation ability, and finally at least 1 ⁇ 10 5 cells or more from one cell. It can grow.
- the medium used in the present invention preferably contains at least one cytokine selected from bFGF, EGF, TGF, NGF, and Wnt3a.
- the medium may contain a serum substitute such as KSR.
- a serum substitute such as KSR.
- 1 or 2 or more chosen from retinoic acid, (beta) mercaptoethanol, sodium pyruvate, and ascorbic acid may be included.
- Adhesion culture is performed using a culture vessel coated with collagen, laminin, fibronectin, Matrigel TM , poly-L-ornithine / laminin, or poly-D-lysine (PDL).
- collagen laminin, fibronectin, Matrigel TM , poly-L-ornithine / laminin, or poly-D-lysine (PDL).
- corneal endothelial progenitor cells can be cultured for a long time in an undifferentiated state with high proliferation ability.
- the culture is continued for 7 days or more.
- the origin of the corneal endothelial cell tissue used is not limited, it is preferably a human origin.
- Corneal endothelial progenitor cells can be obtained by isolating a p75 positive cell group from the cell group cultured by the above method.
- the present invention also provides a method for preparing corneal endothelial cells, characterized by inducing differentiation of the corneal endothelial progenitor cell group prepared by the above method into a corneal endothelial cell group.
- Induction of differentiation into corneal endothelial cell population can be performed by using a medium containing serum-free medium, or serum containing the TGF- ⁇ 2.
- Differentiation induction into corneal endothelial cells is performed by using a culture dish (vessel) coated with collagen, laminin, fibronectin, Matrigel TM , poly-L-ornithine / laminin (PLO / LM), poly-D-lysine (PDL), or the like.
- a culture dish vessel coated with collagen, laminin, fibronectin, Matrigel TM , poly-L-ornithine / laminin (PLO / LM), poly-D-lysine (PDL), or the like.
- corneal endothelial precursor cells may be cultured on a polymer membrane to induce differentiation into corneal endothelial cells.
- the present invention further provides a method for producing a corneal endothelial cell sheet by culturing a corneal endothelial precursor cell group or a corneal endothelial cell group prepared by the method of the present invention using a polymer membrane as a support.
- Polymer membranes used include, for example, glycosaminoglycans including chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, heparin, and keratan sulfate; atelocollagen, alkali-treated collagen, and gelatin; keratin; proteoglycan; alginic acid, chitosan, And hyaluronic acid; polyamino acid (polylactic acid); cellulose; (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, vinyl ether derivatives, and copolymers containing these A polymer containing any one or two or more selected from the group consisting of functional polymers.
- glycosaminoglycans including chondroitin sulfate, dermatan sulfate, hyaluronic acid, hepara
- cells derived from corneal endothelial tissue can be grown in a more undifferentiated state (while maintaining a p75 positive state).
- the corneal endothelial progenitor cell group obtained in the present invention has a high proliferative ability and can therefore be subcultured over a long period of time, whereby a large amount of corneal endothelial progenitor cell group can be prepared.
- the corneal endothelial progenitor cell group obtained in the present invention can be induced to differentiate into mature corneal endothelial cells by culturing under appropriate conditions. Therefore, according to the present invention, it is possible to supply a stable corneal endothelial cell source in the long term, and it is possible to solve the problem of serious donor shortage (10 million waiting patients) in corneal regenerative medicine.
- FIG. 1 shows cells (A: primary culture 14 days, B: primary culture 21 days) obtained from corneal endothelial tissue by the method of the present invention.
- FIG. 2 shows expression of neural crest markers p75, SOX9, FOXC2 and corneal endothelial marker N-cadherin in corneal endothelial progenitor cells obtained by the method of the present invention. From the left of each graph, corneal endothelial precursor cells obtained by the method of the present invention, cells obtained by a known method, and in vivo corneal endothelial cells are shown.
- FIG. 3 is a graph comparing the expression level of p75 in corneal endothelial progenitor cells obtained by the method of the present invention with other cells.
- FIG. 4 shows the proliferation ability of corneal endothelial progenitor cells obtained by the method of the present invention.
- A Photograph after 2 days of culture of primary cultured cells (left) and first passage cells (right).
- B Expression of proliferation marker Ki-67 (left: stained image of Ki-67, right: stained image of Ki-67 and nuclear stain).
- C Proliferation capacity (growth curve) of each passage cell.
- FIG. 5A shows images of corneal endothelial cells differentiated from corneal endothelial progenitor cells obtained by the method of the present invention (left: corneal endothelial progenitor cells obtained by the method of the present invention, right: normal cultured corneal endothelial cells).
- FIG. 5B shows expression of corneal endothelial marker type VIII collagen (COL8A2) (from the left of the graph, corneal endothelial progenitor cells, corneal endothelial cells differentiated from corneal endothelial progenitor cells, cells obtained by a known method, in vivo Corneal endothelial cells).
- FIG. 6 shows the analysis results of the corneal endothelial cell sheet prepared on the atelocollagen sheet (A: corneal endothelial cell sheet prepared on the atelocollagen sheet, B: position scanning image, C: alizarin stained image, D: Na + / K + -ATPase expression, E: ZO-1 expression, F: N-cadherin expression).
- A corneal endothelial cell sheet prepared on the atelocollagen sheet
- B position scanning image
- C alizarin stained image
- D Na + / K + -ATPase expression
- E ZO-1 expression
- F N-cadherin expression
- FIG. 7 shows the results of analyzing the pumping function of the corneal endothelial cell sheet using an in-vitro evaluation system (using-chamber system) (A: time-dependent change in short-circuit current, B: corneal endothelial cells differentiated from corneal endothelial progenitor cells ( Comparison of left) and cultured corneal endothelium (right)).
- FIG. 8 shows transplantation of a corneal endothelial cell sheet to a rabbit bullous keratopathy model, and transparency of the cornea (A: the upper group is a transplant group, the lower stage is a control group. The results of verifying improvement in corneal thickness (B:- ⁇ -transplant group,- ⁇ -control group) are shown.
- Corneal Endothelial Cell The cornea has a three-layer structure from the surface: a corneal epithelial layer, a corneal stroma layer, and a corneal endothelial layer. “Corneal endothelial cells” are a group of cells constituting the innermost layer of the cornea and do not regenerate in vivo when damaged. A “corneal endothelial cell” is a cell derived from a neural crest, has a paving stone shape, and is characterized by expression of type VIII collagen, ZO-1, Na + / K + ATPase, etc., which are differentiation markers of corneal endothelial cells.
- Corneal endothelial progenitor cells mean undifferentiated cells that can differentiate into corneal endothelium. “Corneal endothelial progenitor cells” are cells having a dendritic morphology and high proliferation ability. Further, in the initial stage of culture (proliferation), the differentiation marker is not expressed, and is characterized by the expression of p75, which is a specific marker for undifferentiated cells such as neural crest stem cells.
- the “corneal endothelial progenitor cells” obtained in the present invention may also have properties as stem cells (multipotency and self-renewal ability). In that sense, “corneal endothelial progenitor cells” can also be described as “corneal endothelial stem cells / progenitor cells”.
- Serum-free medium used in the present invention means a medium that does not contain serum components of other species.
- the serum-free medium has no fear of infection by pathogens derived from animal serum, and the obtained cells and cultures can be safely applied to clinical applications. Therefore, in consideration of utilization of the corneal endothelial cells obtained in the present invention for regenerative medicine and the like, it is preferable to use a serum-free medium as the medium.
- the serum-free medium may contain an artificial serum substitute described later.
- serum substitute used in the present invention means an artificial serum substitute that can be used in place of serum.
- KSR knockout serum replacement: invitrogen (GIBCO) Manufactured
- albumin such as lipid-rich albumin, transferrin, fatty acid, insulin, collagen precursor, trace element, 2-mercaptoethanol, 3′thiolglycerol, or equivalents thereof.
- Cytokines is a general term for proteinaceous factors that are released from cells and mediate various cell-cell interactions. In the present invention, cytokines are added to the medium for the purpose of promoting cell proliferation and differentiation induction.
- bFGF basic fibroblast growth factor
- IFN ⁇ , ⁇ , ⁇ interleukins
- VEGF vascular endothelial growth factor
- TPO thrombopoietin
- G-CSF granules Sphere colony-stimulating factor
- Macrop macrophage colony-stimulating factor
- a protein (cell marker) specifically expressed in each cell type is used.
- the corneal endothelial progenitor cell group according to the present invention is specified by p75, FOXC2, SOX9, N-cadherin positive in the initial stage of culture. These markers can be maintained for a certain period in the culture system of the present invention, but disappear gradually as the growth proceeds.
- corneal endothelial progenitor cells do not express ki-67 at the initial stage of culture, but highly express ki-67 during proliferation.
- a cell group containing corneal endothelial cells induced to differentiate is identified by positive type VIII collagen.
- the nerve growth factor receptor is a receptor for neurotrophins
- p75 is known as a low affinity neurotrophin receptor and is a marker of migrating neural crest cells. p75 is expressed only in a small number of stem cells existing in adults including neural crest stem cells, and is one of the most reliable markers as an undifferentiated index.
- FOXC2 Forward box protein C2
- SOX9 SOX10
- AP2 ⁇ AP2 ⁇
- snail, slug PITX2, FOXC1, and the like are also known.
- N-cadherin is a protein belonging to the calcium-dependent cell adhesion molecule, and plays an important role in cell adhesion through the interaction of the same type of cadherin and the binding to the actin cytoskeleton via catenin, and may be involved in the developmental differentiation stage.
- N-cadherin is a mesenchymal marker expressed in various tissues such as nerve, cardiac muscle, skeletal muscle, vascular endothelium and corneal endothelium.
- Type VIII collagen is a non-fibrous collagen that is highly expressed in tissues with active morphogenesis and is known as a marker for differentiated corneal endothelial cells.
- examples of the marker for differentiated corneal endothelial cells include ZO-1.
- the method of the present invention is mainly characterized by (1) serum-free medium, (2) low-density culture, and (3) adhesion culture.
- serum-free medium In the method of the present invention, cells are cultured using a serum-free medium that does not contain serum.
- a serum-free medium that does not contain serum.
- As the basic medium DMEM medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, Dulbecco MEM medium, Ham medium, Any medium that can be used for culturing animal cells, such as RPMI 1640 medium, Fischer's medium, McCoy's medium, Williams E medium, and mixed medium thereof, can be used.
- the medium used in the culture method of the present invention is prepared by adding various nutrient sources necessary for the maintenance and growth of cells and components necessary for differentiation induction to the above basic medium.
- the medium preferably contains cytokines or serum substitutes that promote cell growth.
- cytokines added to the medium include basic fibroblast growth factor (bFGF), transforming growth factor ⁇ (transforming factor- ⁇ : TGF- ⁇ ), and interferons (IFN ⁇ , ⁇ ). , ⁇ ), interleukins, vascular endothelial growth factor (VEGF), thrombopoietin (TPO), granulocyte colony-stimulating factor (CLO), granulocyte colony-stimulating factor (CS).
- Stimulation factor Macrophage-policy stimul ting factor; M-CSF
- nerve growth factor nerve growth factor (nerve growth factor: NGF), and the like can be given.
- Serum substitutes include albumin (eg, lipid-rich albumin), transferrin, fatty acid, insulin, collagen precursor, trace elements, ⁇ -mercaptoethanol or 3 ′ thiol glycerol, commercially available Knockout Serum Replacement (KSR), Chemically-defined Lipid and concentrated (Gibco) and Glutamax (Gibco).
- albumin eg, lipid-rich albumin
- transferrin fatty acid
- insulin insulin
- collagen precursor e.g., collagen precursor
- trace elements ⁇ -mercaptoethanol or 3 thiol glycerol
- KSR Knockout Serum Replacement
- Gabco Chemically-defined Lipid and concentrated
- Glutamax Gibco
- an amino acid reducing agent such as pyruvic acid or ⁇ -mercaptoethanol, an amino acid, or the like can be added to the medium as necessary.
- carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, dextrin, hydrocarbons such as fatty acids, fats and oils, lecithin, alcohols, ammonium sulfate, ammonium nitrate, Nitrogen sources such as ammonium chloride, urea, sodium nitrate, salt, potassium salt, phosphate, magnesium salt, inorganic salt such as calcium salt, iron salt, manganese salt, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, Sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate and manganese sulfate, various vitamins, and the like can be included.
- carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, dextrin, hydrocarbons such as fatty acids, fats and oils, lecithin, alcohols, ammonium s
- the pH of the medium obtained by blending these components is in the range of 5.5 to 9.0, preferably 6.0 to 8.0, and more preferably 6.5 to 7.5.
- corneal endothelial cells are cultured at a high density of 10,000 to 100,000 cells / cm 2 , or explant culture is performed with the Descemet's membrane. However, in the method of the present invention, at least less than 10000 cells / cm 2 , preferably 5000 cells / cm 2 or less, more preferably 20 to 2000 cells / cm 2 , more preferably 50 to 1000 cells / cm 2 , most preferably 100 to 500 cells / cm 2. Cells are seeded at a low density of about 2 and cultured.
- Adhesive culture In the method of the present invention, a culture dish (vessel) coated with collagen, laminin, fibronectin, Matrigel TM , poly-L-ornithine / laminin (PLO / LM), poly-D-lysine (PDL) or the like. Is used for adhesion culture.
- the culture is performed at 36 ° C. to 38 ° C., preferably 36.5 ° C. to 37.5 ° C. under the conditions of 1% to 25% O 2 and 1% to 15% CO 2 .
- the culture is performed for at least 7 days, preferably 7 to 14 days, changing the medium once every 2 to 3 days.
- the cell shape changes with passage, making it difficult to maintain corneal endothelial function and proliferation ability.
- the cell morphology does not change even when passaged, and long-term culture is possible while maintaining high growth ability. This is because in the method of the present invention, cells derived from corneal endothelial tissue can be grown while maintaining an undifferentiated state. In other words, in the method of the present invention, a more undifferentiated cell group having a high proliferation ability can be selectively proliferated.
- the corneal endothelial progenitor cell group selectively proliferated in the present invention is distinguished from the corneal endothelium-derived cell group cultured by a conventionally known method in that p75 is an undifferentiated marker. Although the expression of p75 gradually disappears as the growth proceeds, these cell groups have a high proliferation ability and can finally grow up to 100,000 times.
- This cell group is obtained by culturing by a conventionally known method also in that it expresses neural crest markers SOX9 and FOXC2 and corneal endothelial marker N-cadherin at the start of culture. It is distinguished from a group of cells derived from the corneal endothelium. The expression of SOX9, FOXC2, and N-cadherin also disappears little by little as the growth proceeds.
- the corneal endothelial progenitor cell group proliferated by the method of the present invention expresses the proliferation marker Ki-67, has a high proliferative capacity, and can finally proliferate from 1 cell to at least 1 ⁇ 10 5 cells or more. It is a group of cells with high proliferation ability. This group of cells can differentiate into corneal endothelial cells under appropriate conditions. In the present invention, this group of cells that can differentiate into corneal endothelial cells is referred to as “corneal endothelial cell progenitor cells (group)”.
- corneal endothelial progenitor cells obtained by the method of the present invention can be differentiated into mature corneal endothelial cells by culturing under appropriate conditions. Can do.
- DMEM medium As basic medium, DMEM medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, Dulbecco MEM Any medium that can be used for culturing animal cells, such as a medium, ham medium, RPMI 1640 medium, Fischer's medium, McCoy's medium, Williams E medium, and mixed medium thereof, can be used.
- a medium, ham medium, RPMI 1640 medium, Fischer's medium, McCoy's medium, Williams E medium, and mixed medium thereof can be used.
- the medium used for inducing differentiation into corneal endothelial cells is composed of the above-mentioned basic medium containing factors that promote differentiation induction into corneal endothelial cells, various nutrients necessary for cell maintenance and proliferation, and various components necessary for induction of differentiation. Made by adding.
- factors that promote differentiation into corneal endothelial cells include TGF- ⁇ 2 , cholera toxin, transferrin, insulin, EGM (Epidmal Growth Factor), serum or serum substitute, KSR (Knockout Serum Replacement), and the like. .
- the induction of differentiation into corneal endothelial cells using the culture medium containing serum-free medium (see JP 2009-268433), or serum such as BSA containing TGF- ⁇ 2.
- antibiotics such as penicillin and streptomycin, amino acid reducing agents such as cytokines, pyruvate and ⁇ -mercaptoethanol, antioxidants such as ascorbic acid, amino acids and the like can be added to the medium as necessary.
- carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, dextrin, hydrocarbons such as fatty acids, fats and oils, lecithin, alcohols, ammonium sulfate, ammonium nitrate, Nitrogen sources such as ammonium chloride, urea, sodium nitrate, salt, potassium salt, phosphate, magnesium salt, inorganic salt such as calcium salt, iron salt, manganese salt, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, Sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate and manganese sulfate, various vitamins, and the like can be included.
- carbon sources such as glycerol, glucose, fructose, sucrose, lactose, honey, starch, dextrin, hydrocarbons such as fatty acids, fats and oils, lecithin, alcohols, ammonium s
- the pH of the medium obtained by blending these components is in the range of 5.5 to 9.0, preferably 6.0 to 8.0, and more preferably 6.5 to 7.5.
- Culture conditions Culture is performed using a culture dish (vessel) coated with collagen, laminin, fibronectin, Matrigel TM , poly-L-ornithine / laminin (PLO / LM), poly-D-lysine (PDL), or the like. 36 to 38 ° C., preferably 36.5 to 37.5 ° C., under the conditions of 1% to 25% O 2 and 1% to 15% CO 2 .
- the culture period is changed at least once every 2 to 3 days, and is at least 7 days, preferably about 1 to 8 weeks.
- Differentiation induction from corneal endothelial progenitor cell groups to mature corneal endothelial cells can be confirmed by changes in morphology (changes to cobblestone-like cells), expression of mature corneal endothelial cell marker type VIII collagen and the like. .
- the corneal endothelial progenitor cells grown by the method of the present invention can be easily isolated (purified) using the surface marker p75 or the like.
- it can be isolated using immunomagnetic beads labeled with an antibody specific to p75, a column on which a p75 antibody is immobilized, and separation by a cell sorter (FACS) using a fluorescently labeled p75 antibody.
- FACS cell sorter
- the corneal endothelial cell group induced to differentiate by the method of the present invention can be isolated using the expression of its specific marker, such as type VIII collagen, as an index, although it is not a surface marker.
- 5.2 Cell preparation for treating corneal endothelial disease
- Differentiated and isolated corneal endothelial progenitor cells by the method of the present invention, and / or corneal endothelial cells induced to differentiate from the cells are corneal endothelial diseases. It can be used as a cell preparation.
- the administration method of the cell preparation of the present invention is not particularly limited, and local transplantation, intravenous administration, and local injection administration by surgical means can be performed depending on the application site.
- the cell preparation of the present invention may contain scaffold materials and components for assisting cell maintenance / proliferation and administration to the affected area, and other pharmaceutically acceptable carriers.
- Components necessary for cell maintenance / proliferation include media components such as carbon sources, nitrogen sources, vitamins, minerals, salts, various cytokines, and extracellular matrix preparations such as Matrigel TM .
- the cell preparation of the present invention may contain scaffold materials and components for assisting cell maintenance / proliferation and administration to the affected area, and other pharmaceutically acceptable carriers.
- Components necessary for cell maintenance / proliferation include media components such as carbon sources, nitrogen sources, vitamins, minerals, salts, various cytokines, and extracellular matrix preparations such as Matrigel TM .
- scaffold materials and components that assist administration to the affected area include biodegradable polymers; for example, collagen, polylactic acid, hyaluronic acid, cellulose, and derivatives thereof, and a complex composed of two or more thereof, an aqueous solution for injection;
- biodegradable polymers for example, collagen, polylactic acid, hyaluronic acid, cellulose, and derivatives thereof, and a complex composed of two or more thereof, an aqueous solution for injection
- examples include physiological saline, medium, physiological buffer such as PBS, and isotonic solutions (eg, D-sorbitol, D-mannose, D-mannitol, sodium chloride) containing glucose and other adjuvants.
- An auxiliary agent such as alcohol, specifically ethanol, polyalcohol such as propylene glycol, polyethylene glycol, nonionic surfactant such as polysorbate 80, HCO-50, etc. may be used.
- organic solvents polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose as necessary , Ethyl cellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, It may contain a buffer, an emulsifier, a suspension, a soothing agent, a stabilizer and the like.
- a purified antibody is dissolved in a solvent such as physiological saline, buffer solution, glucose solution, etc., and an adsorption inhibitor such as Tween 80, Tween 20, gelatin or the like is added thereto.
- a solvent such as physiological saline, buffer solution, glucose solution, etc.
- an adsorption inhibitor such as Tween 80, Tween 20, gelatin or the like is added thereto.
- Tween 80, Tween 20, gelatin or the like is added thereto.
- a corneal endothelial cell sheet can be prepared by culturing a group of corneal endothelial cells obtained by the method described in 3 above on an appropriate polymer membrane (carrier).
- the polymer membrane used is collagen, atelocollagen, alkali-treated collagen, gelatin, keratin, hyaluronic acid, glycosaminoglycan (chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, heparin, keratan sulfate), proteoglycan, alginic acid, chitosan Biopolymers such as polyamino acids (polylactic acid) and cellulose, or (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, vinyl ether derivatives, copolymers thereof, etc. And a temperature-responsive polymer.
- corneal endothelial dysfunction including bullous keratopathy, corneal dystrophy, developmental glaucoma, Rieger malformation, congenital hereditary corneal endothelial dystrophy , Limbal delmoid, sclera, corneal abnormalities such as keratoconus and perosidic corneal degeneration, corneal scar, corneal infiltration, corneal deposition, corneal edema, corneal ulcer, ocular trauma including chemicals and heat, keratitis , Eye diseases such as corneal degeneration and corneal infection, neuroblastoma, Hirschsprung disease, Waadenburg syndrome, localized baldness Recklinghausen disease.
- Matrigel TM hESC-qualified Matrix (BD) used for culturing corneal endothelial progenitor cells Matrigel TM thawed on ice or at 4 ° C. was diluted 30-fold with cold DMEM / F12 medium (Invitrogen), added to the culture dish, and incubated at 37 ° C. for 1 hour to coat the culture dish.
- DMEM / F12 medium Invitrogen
- Laminin 511 (Veritas) Laminin 511 was diluted to 20 ⁇ g / ml with PBS, added to the culture dish, and incubated at 37 ° C. for 2 hours to coat the culture dish. After washing twice with PBS and twice with the medium used, the medium was added.
- Y-27632 (Wako, apoptosis inhibitor) was added to cells cultured until passage subconfluence of corneal endothelial progenitor cells to a final concentration of 10 ⁇ M, left at 37 ° C. for 60 minutes, and washed once with PBS Then, 0.25 ml of Stem Pro Accutase (Invitrogen, cell lysate) was added and left at room temperature for 2-5 minutes.
- FNC coating mix (AthenaES) FNC coating mix (a coating agent containing fibronectin and type I collagen) was added to the culture dish and allowed to stand at room temperature for 30 seconds.
- corneal endothelial progenitor cells Differentiation from corneal endothelial progenitor cells to mature corneal endothelial cells Y-27632 (Wako) was added to the corneal endothelial progenitor cells cultured by the above method to a final concentration of 10 ⁇ M, treated at 37 ° C. for 60 minutes, and PBS After washing once, 0.25 ml of Stem Pro Accutase (Invitrogen, cell lysate) was added and incubated at room temperature for 2-5 minutes.
- Stem Pro Accutase Invitrogen, cell lysate
- telocollagen sheet coated with FNC coating mix (AteloCell, Koken) The cells were seeded on top (seed density: 3,000-5,000 cells / mm 2 ) and cultured while changing the medium once every two to three days. Cells having a corneal endothelium-like (cobblestone) morphology appeared from 1 week to 4 weeks.
- DMEM low-glucose Nikken Biomedical Research Institute, Basic Medium
- FBS Nippon Bioserum
- Penicillin-Streptomycin Invitrogen
- 2ng / ml bFGF Invitrogen
- Corneal endothelial cells by a known method
- cells were isolated from corneal tissue and cultured according to a known method (supra, Yokoo et al., IVOS, 2005). That is, the isolated corneal endothelial cells were seeded on a non-adhesive culture dish using a medium containing DMEM / F12, B27 supplement, 40 ng / ml bFGF, and 20 ng / ml EGF. Once every two days, 40 ng / ml bFGF and 20 ng / ml EGF were added and cultured for a total of 10 days.
- RNA extraction RNA was extracted from the cells using RNeasy Micro Plus kit (QIAGEN).
- cDNA Synthesis cDNA was prepared from the RNA obtained in the previous section using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen).
- the corneal endothelial progenitor cells or corneal endothelial cells prepared by the method described above were fixed with 4% paraformaldehyde (Wako) at room temperature for 30 minutes or cold methanol (Wako) at ⁇ 30 ° C. for 30 minutes.
- the plate was washed twice with a Tris buffer solution (TBS, TaKaRa) and treated with 5% NST (see Table 2 for the composition) for 1 hour at room temperature.
- the primary antibody was adjusted to the optimum concentration with 1% NST, incubated overnight at 4 ° C. (see Table 3), and washed 3 times with TBS.
- a secondary antibody Invitrogen, AlexaFluor 488 or 568 conjugated, anti-IgG antibody was diluted 200-fold with 1% NST, and incubated at room temperature for 2 hours.
- Alizarin staining Alizarin Red S (Wako) was adjusted to 0.9% NaCl (pH 4.2). The cells were stained with a staining solution for 5 minutes, then fixed with 4% paraformaldehyde, and observed under a microscope.
- the pump function was measured using a Ussing chamber. Induced corneal endothelial cell sheets cultured on atelocollagen were mixed with Krebs-Ringer solution (120.7 mM NaCl, 24 mM NaHCO 2 , 4.6 mM KCl, 0.7 mM Na 2 HPO 4 , 0.5 mM MgCl 2 , 10 mM glucose, pH 7.4). ).
- the Ussing chamber system was manufactured by WPI. Short-circuit current was calculated by adding 1 mM ouabain (Sigma) as Na + / K + -ATPase inhibitor.
- Rabbit corneal endothelium disorder model was performed by removing the corneal endothelium from one eye of the rabbit by the cryo method. One week after that, the induced corneal endothelium sheet was adhered to the corneal endothelium surface by corneal endothelium transplantation (DAESK), and air was injected to make it adhere. The corneal thickness was measured with a pachymeter (SP-100, Tomey) over time, and the anterior segment image was observed with a slit lamp (AIP-20, Topcon).
- Cells obtained by the method of the present invention Cells isolated from human corneal endothelial tissue (number of endothelial cells: about 1 to 4 ⁇ 10 4 cells) are cultured under 50-1000 cells / cm 2 / serum free. Thus, the appearance of colonies was observed after culturing for about 7 to 14 days (appearance rate: about 0.1%: about 10 to 40 cells per eye).
- the morphology of the cells grown from the corneal endothelial tissue by the method of the present invention was very similar to that of neural crest cells derived from iPS cells, suggesting the possibility of being corneal endothelial progenitor cells (FIG. 1).
- Corneal progenitor progenitor cells obtained by the method of the present invention are derived from the development of corneal endothelium as compared with endothelial cells cultured by known methods and in vivo cells. Expression of neural crest markers p75, SOX9, FOXC2 and N-cadherin, which is one of corneal endothelial markers, was increased (FIG. 2).
- corneal endothelial cells differentiated from the corneal endothelial progenitor cells obtained by the method of the present invention have a cobblestone-like cell morphology (Fig. 5A) and are corneal endothelial markers. Increased expression of type VIII collagen (COL8A2) was observed (FIG. 5B). From this, it was confirmed that the corneal endothelial progenitor cells obtained by the method of the present invention can be differentiated into corneal endothelial cells.
- Atelocollagen sheet is a highly safe medical collagen sheet and is one of carrier candidates for transplantation of corneal endothelium.
- a transparent corneal endothelial cell sheet could be prepared (FIG. 6A), and a cobblestone-like cell morphology was exhibited (FIGS. 6B and C).
- induced corneal endothelial cell sheet In corneal endothelial cells replated on an atelocollagen sheet, corneal endothelial markers Na + / K + -ATPase, ZO, as well as corneal endothelial cells induced to differentiate from corneal endothelial precursor cells. -1 and N-cadherin expression was observed (FIGS. 6D, E, F). From this, it was shown that the induced corneal endothelial cell sheet contains mature corneal endothelial cells and can be used as a material for regenerative medicine.
- corneal endothelial tissue can be grown in a more undifferentiated state (positive for p75, FOXC2, and SOX9).
- the resulting corneal endothelial progenitor cells had a high proliferative ability and could theoretically produce about 330 corneal endothelial cell sheets from one donor.
- the cells were confirmed to be corneal endothelial precursor cells having the ability to differentiate into mature corneal endothelial cells.
- cells derived from corneal endothelial tissue can be grown while maintaining an undifferentiated state.
- more undifferentiated corneal endothelial progenitor cells with high proliferation ability can be selectively proliferated from cells derived from corneal endothelial tissue.
- the corneal endothelial progenitor cell group obtained in the present invention can be induced to differentiate into mature corneal endothelial cells by culturing under appropriate conditions. Therefore, the present invention can be used as a basic technology for regenerative medicine for bullous keratopathy and the like.
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Abstract
Description
以下、本発明にかかる用語について説明する。
(1)角膜内皮細胞
角膜は、表面から、角膜上皮層、角膜実質層、角膜内皮層の3層構造をしている。「角膜内皮細胞」は、この角膜の一番内側の層を構成する細胞群で、障害を受けるとin vivoでは再生しない。「角膜内皮細胞」は神経堤に由来する細胞で、敷石状の形態を有し、角膜内皮細胞の分化マーカーであるタイプVIIIコラーゲン、ZO−1、Na+/K+ ATPase等の発現によって特徴づけられる。
本発明にかかる「角膜内皮前駆細胞」とは、角膜内皮に分化可能な未分化細胞を意味する。「角膜内皮前駆細胞」は、樹状形態を有する細胞で、高い増殖能を有する。また、培養(増殖)初期段階では、分化マーカーを発現しておらず、神経堤幹細胞等の未分化細胞特異的なマーカーであるp75の発現によって特徴づけられる。本発明で得られる「角膜内皮前駆細胞」は、幹細胞としての性質(多分化能と自己複製能)も有する可能性がある。その意味では、「角膜内皮前駆細胞」は「「角膜内皮幹細胞・前駆細胞」と記載することもできる。
本発明で用いられる「無血清培地」とは、他種血清成分を含まない培地を意味する。無血清培地は、動物血清に由来する病原体による感染の恐れがなく、得られた細胞や培養物は安全に、臨床応用に適用することができる。それゆえ、本発明で得られる角膜内皮細胞の再生医療等への利用を考慮した場合、培地としては無血清培地を用いることが好ましい。なお、無血清培地は、後述する工血清代替物を含んでいてもよい。
本発明で用いられる「血清代替物」とは、血清に代替して使用可能な人工血清代替物を意味し、例えば、前述したKSR(knockout serum replacement:invitrogen社(GIBCO)製)、脂質リッチアルブミン等のアルブミン、トランスフェリン、脂肪酸、インスリン、コラーゲン前駆体、微量元素、2−メルカプトエタノールや3’チオールグリセロール、あるいはこれらの均等物を挙げられる。
「サイトカイン」とは、細胞から放出され、種々の細胞間相互作用を媒介するタンパク質性因子の総称である。本発明においては、細胞の増殖と分化誘導を促進させる目的で、培地にサイトカインを添加する。
本発明では、角膜内皮前駆細胞、及び、分化誘導された角膜内皮細胞を同定するために、各細胞種に特異的に発現しているタンパク質(細胞マーカー)を利用する。具体的には、本発明にかかる角膜内皮前駆細胞群は、培養初期段階においては、p75、FOXC2、SOX9、N−cadherin陽性によって特定される。これらのマーカーは、本発明の培養系において一定期間維持することが可能であるが、増殖が進むにつれて少しずつ消失する。一方で、角膜内皮前駆細胞は培養初期にはki−67を発現していないが、増殖時にはki−67を高発現するようになる。一方分化誘導された角膜内皮細胞を含む細胞群はタイプVIIIコラーゲン陽性によって特定される。
2.1 細胞の単離
まず、常法にしたがい、単離された角膜内皮組織をトリプシン、コラゲナーゼ等の酵素で処理して、細胞を単離させる。単離させた細胞は、DMEM等の基本培地に懸濁させ、遠心して組織断片を除去する。こうして調製した角膜内皮組織由来の細胞群を以下の手順で培養を行う。
本発明の方法は、(1)無血清培地、(2)低密度培養、(3)接着培養の3つを主たる特徴とする。
本発明の方法では、血清を含まない無血清培地を用いて細胞を培養する。基本培地としては、DMEM培地、BME培地、BGJb培地、CMRL 1066培地、Glasgow MEM培地、Improved MEM Zinc Option培地、IMDM培地、Medium 199培地、Eagle MEM培地、α MEM培地、Dulbecco MEM培地、ハム培地、RPMI 1640培地、Fischer’s培地、McCoy’s培地、ウイリアムスE培地、及びこれらの混合培地など、動物細胞の培養に用いることのできる培地であればいずれも用いることができる。
一般に角膜内皮細胞の培養は、10,000~100,000cells/cm2の高密度で細胞を播種、またはデスメ膜ごと外植片培養を行う。しかし、本発明の方法では少なくとも10000cells/cm2未満、好ましくは5000cells/cm2以下、より好ましくは20~2000cells/cm2、さらに好ましくは50−1000cells/cm2、もっとも好ましくは100~500cells/cm2程度の低密度で細胞を播種して培養を行う。
本発明の方法では、コラーゲン、ラミニン、フィブロネクチン、マトリゲルTM、ポリ−L−オルニチン/ラミニン(PLO/LM)又はポリ−D−リジン(PDL)等でコーティングした培養皿(器)を用いて、接着培養を行う。
血清を含む培地を用いた従来の方法では、継代するにつれて細胞形態が変化し、角膜内皮機能や増殖能を維持することが困難になる。しかしながら、本発明の方法では、継代しても細胞形態は変化せず、高い増殖能を維持したまま、長期間の培養が可能となる。これは、本発明の方法では、角膜内皮組織由来の細胞を、未分化な状態を維持したまま増殖させることができるからである。換言すれば、本発明の方法では、増殖能の高い、より未分化な細胞群が選択的に増殖させることができる。
上記のとおり、本発明の方法によって得られた角膜内皮前駆細胞群は、適当な条件下で培養することにより、成熟した角膜内皮細胞に分化させることができる。
基本培地としては、DMEM培地、BME培地、BGJb培地、CMRL 1066培地、Glasgow MEM培地、Improved MEM Zinc Option培地、IMDM培地、Medium 199培地、Eagle MEM培地、α MEM培地、Dulbecco MEM培地、ハム培地、RPMI 1640培地、Fischer’s培地、McCoy’s培地、ウイリアムスE培地、及びこれらの混合培地など、動物細胞の培養に用いることのできる培地であればいずれも用いることができる。
培養は、コラーゲン、ラミニン、フィブロネクチン、マトリゲルTM、ポリ−L−オルニチン/ラミニン(PLO/LM)又はポリ−D−リジン(PDL)等でコーティングした培養皿(器)を用いて、36℃~38℃、好ましくは36.5℃~37.5℃で、1%~25% O2、1%~15% CO2の条件下で行われる。培養期間は、2~3日に1回培地を交換し、少なくとも7日、好ましくは1週間~8週間程度である。
本発明の方法によって増殖された角膜内皮前駆細胞群は、表面マーカーであるp75等を利用して、簡便に単離(純化)することができる。例えば、p75に特異的な抗体で標識された免疫磁気ビーズ、p75抗体を固相化したカラム、蛍光標識されたp75抗体を用いたセルソーター(FACS)による分離を用いて単離することができる。
5.1 培養物
本発明の方法によって得られた角膜内皮前駆細胞群、及び/又は前記細胞群から分化誘導された角膜内皮細胞群を含む培養物は、研究、再生医療あるいは後述する細胞製剤の原料として利用することができる。
本発明の方法によって、分化誘導され、単離された角膜内皮前駆細胞群、及び/又は前記細胞群から分化誘導された角膜内皮細胞群は、角膜内皮疾患用細胞製剤として利用できる。
(1)キャリア上での培養
本発明の方法で得られた角膜内皮前駆細胞群を適当な高分子膜(キャリア)上で培養し、角膜内皮細胞に分化誘導させることにより、あるいは、上記3に記載した方法で得られた角膜内皮細胞群を適当な高分子膜(キャリア)上で培養することにより角膜内皮細胞シートを作製することができる。
1.角膜内皮前駆細胞の培養・維持
角膜内皮前駆細胞の樹立・維持培地
・DMEM/F−12(Invitrogen社、基礎培地)
・20% KnockOUT Serum Replacement(Invitrogen社、血清代替物)
・2mM L−グルタミン(Invitrogen社)
・1%非必須アミノ酸(Invitrogen社)
・100μM 2−メルカプトエタノール(Invitrogen社)
・4ng/ml basic−FGF(Wako社)
・マトリゲルTM hESC−qualified Matrix(BD社)
氷上又は4℃で融解したマトリゲルTMを冷DMEM/F12培地(Invitrogen社)で30倍希釈し、培養皿に添加し、37℃、1時間インキュベートして培養皿をコーティングした。
・ラミニン511(ベリタス社)
ラミニン511をPBSで20μg/mlに希釈し、培養皿に添加し、37℃、2時間インキュベートして培養皿をコーティングした。PBSで2回、使用する培地で2回洗浄した後、培地を加えた。
ヒト角膜内皮組織をデスメ膜ごと剥離し、10μM Y−27632(Wako社,ROCK阻害剤;アポトーシス阻害剤)を含むDMEM(Invitrogen社)の入った3.5cm培養皿に移し、37℃で30分維持した。
サブコンフルエントまで培養した細胞に、Y−27632(Wako社、アポトーシス阻害剤)を終濃度10μMになるように添加し、37℃で60分放置し、PBSで1回洗浄したのち、0.25mlのStem Pro Accutase(Invitrogen社,細胞乖離液)を加え、室温で2~5分放置した。
角膜内皮前駆細胞から成熟角膜内皮細胞への分化誘導培地
・DMEM low−glucose(日研生物医学研究所)
・10% FBS(日本バイオシーラム社)
・2mM L−グルタミン(Invitrogen社)
・1% Penicillin−Streptomycin(Invitrogen社)
・FNC coating mix(AthenaES社)
FNC coating mix(フィブロネクチンとタイプIコラーゲンを含むコーティング剤)を培養皿に加え、室温、30秒放置した。
上記方法で培養した角膜内皮前駆細胞にY−27632(Wako社)を終濃度10μMになるように添加し、37℃で60分処理し、PBSで1回洗浄したのち、0.25mlのStem Pro Accutase(Invitrogen社,細胞乖離液)を加え、室温で2~5分間インキュベーションした。
・DMEM low−glucose(日研生物医学研究所,基礎培地)
・10% FBS(日本バイオシーラム社)
・2mM L−グルタミン(Invitrogen社)
・1% Penicillin−Streptomycin(Invitrogen社)
・2ng/ml bFGF(Invitrogen社)
1と同様にして、角膜組織から細胞を単離し、公知の方法にしたがい培養を行った(前掲、Yokooら,IVOS,2005)。すなわち、単離した角膜内皮細胞をDMEM/F12,B27 supplement,40ng/ml bFGF,20ng/ml EGFを含む培地を使用し、非接着性培養皿上に播種した。2日に1回40ng/ml bFGF,20ng/ml EGFを追加し、計10日間培養した。
RNA抽出
RNeasy Micro Plus kit(QIAGEN社)を用いて、細胞からRNAを抽出した。
SuperScript III First−Strand Synthesis SuperMix for qRT−PCR(Invitrogen社)を用いて、前項で得たRNAからcDNAを調製した。
TaqMan Fast Universal PCR Master Mix(Applied Biosystems社)と、表1記載のTaqMan probe(Applied Biosystems社)を用いて、7500 FastリアルタイムPCRシステム(Applied Biosystems社)によりリアルタイムPCRを行った。データ解析は、GAPDHを内因性コントロールとして、ΔΔCt法により行った。
上記した方法で調製した角膜内皮前駆細胞又は角膜内皮細胞を、4%パラホルムアルデヒド(Wako社)で室温30分又は冷メタノール(Wako社)で−30℃、30分で固定した。トリス緩衝溶液(TBS,TaKaRa社)で2回洗浄し、5% NST(組成は表2参照)で室温1時間処理した。
アリザリンレッドS(Wako社)を0.9%NaCl(pH4.2)に調整した。細胞を染色液で5分間染色したのち、4%パラホルムアルデヒドにより固定し、顕微鏡下で観察した。
ポンプ機能はUssing chamberを用いて測定した。アテロコラーゲン上で培養した誘導角膜内皮細胞シートをKrebs−Ringer溶液(120.7mM NaCl,24mM NaHCO2,4.6mM KCl,0.7mM Na2HPO4,0.5mM MgCl2,10mM glucose,pH7.4)でインキュベートした。Ussing chamberシステムはWPI社製のものを用いた。Na+/K+ −ATPase阻害剤である1mMウワバイン(Sigma社)を加え、短絡電流を算出した。
家兎角膜内皮障害モデルは家兎の片眼をクライオ法によって角膜内皮を脱落させた。その1週間後、誘導角膜内皮シートの角膜内皮移植術(DAESK)によって角膜内皮面に接着させ、空気を注入して密着させた。角膜厚は経時的にパキメーター(SP−100,Tomey社)によって測定し、前眼部像はスリットランプによって観察した(AIP−20、トプコン社)。
(1)本発明の方法で得られた細胞
ヒト角膜内皮組織(内皮細胞数:約1~4×104細胞)から単離した細胞を50−1000cells/cm2/無血清下で培養することにより、約7~14日間培養で、コロニーの出現が認められた(出現率:約0.1%:1眼あたりに約10~40細胞存在)。
本発明の方法で得られた角膜内皮前駆細胞は公知の方法で培養した内皮細胞や、in vivoの細胞と比較して、角膜内皮の発生に由来する神経堤マーカーp75、SOX9、FOXC2と、角膜内皮マーカーの一つであるN−cadherinの発現が亢進していた(図2)。
角膜内皮組織から公知の方法で得られた細胞や生体から単離しただけの細胞(in vivo)ではp75の発現が認められなかったが、本発明の方法で得られた角膜内皮幹細胞・前駆細胞では、p75の発現が認められた(図3)。p75(CD271/NGFR)は、神経堤幹細胞を含めた、生体内に少数存在する幹細胞にのみ発現するマーカーで、未分化性の指標である。上記の結果は、本発明の方法では、角膜内皮前駆細胞をより未分化な状態で増殖させることが可能であることを示す。
本発明の方法で得られた角膜内皮前駆細胞では、増殖マーカーの発現が認められた(図4B)。また、継代を繰り返すことによって単一細胞から最大約108個に増幅可能であった(図4C)。このことは、1ドナーから理論上最大、細胞シート8×104枚が作製可能であることを示す。これに対し、公知の方法で得た細胞では、細胞形態が変化し、増殖能が低下するため、継代は困難である(前掲、Yokooら,IVOS,2005)。
本発明の方法で得られた角膜内皮前駆細胞から分化した角膜内皮細胞は敷石状の細胞形態を呈し(図5A)、角膜内皮マーカーであるタイプVIIIコラーゲン(COL8A2)の発現の亢進が認められた(図5B)。このことから、本発明の方法で得られた角膜内皮前駆細胞は角膜内皮細胞に分化可能であることが確認された。
アテロコラーゲンシートは安全性の高い医療用のコラーゲンシートで、角膜内皮の移植用キャリア候補の一つである。角膜内皮前駆細胞から誘導された角膜内皮細胞はアテロコラーゲンシート上に播種すると透明な角膜内皮細胞シートが作製でき(図6A)、敷石状の細胞形態を呈した(図6B、C)。
アテロコラーゲンシート上に再播種した角膜内皮細胞では、角膜内皮前駆細胞から分化誘導した角膜内皮細胞と同様に、角膜内皮マーカーであるNa+/K+ −ATPase、ZO−1及びN−Cadherinの発現が認められた(図6D、E、F)。このことから、誘導角膜内皮細胞シートは成熟角膜内皮細胞を含み、再生医療用材料として使用可能であることが示された。
本発明で得られた誘導角膜内皮細胞シートをUssing chamberを用いてポンプ機能を解析した(図7A)。その結果、培養角膜内皮細胞と比較して同程度であった(図7B)。
本発明で得られた誘導角膜内皮細胞シートを家兎角膜内皮障害モデルへ移植するとアテロコラーゲンのみを移植したコントロール群と比較して、移植後2週間目より顕著な角膜厚の減少が認められた(図8A、B)。このことから、誘導角膜内皮細胞シートは成熟角膜内皮細胞を含み、再生医療用材料として使用可能であることが示された。
本発明の方法により、角膜内皮組織由来の細胞をより未分化な状態(p75及びFOXC2、SOX9 陽性)で増殖させることが可能なことが確認された。得られる角膜内皮前駆細胞は、高い増殖能を有し、1ドナーから理論上、約330枚の角膜内皮細胞シート作製可能であった。当該細胞は成熟角膜内皮細胞への分化能を有する角膜内皮前駆細胞であることが確認された。
Claims (15)
- 角膜内皮細胞組織から単離した細胞群を、低密度で、無血清培地を用いて接着培養して角膜内皮前駆細胞を得ることを特徴とする、角膜内皮前駆細胞の調製方法。
- 細胞群を5000cells/cm2以下の密度で播種して培養することを特徴とする、請求項1記載の方法。
- 細胞群を20~2000cells/cm2の密度で播種して培養することを特徴とする、請求項1記載の方法。
- 少なくとも7日以上培養することを特徴とする、請求項1~3のいずれか1項に記載の方法。
- 得られる角膜内皮前駆細胞がKi−67陽性である、請求項1~4のいずれか1項に記載の方法。
- 増殖される角膜内皮前駆細胞がp75陽性である、請求項1~5のいずれか1項に記載の方法。
- 増殖される角膜内皮前駆細胞が、FOXC2、SOX9、及びN−cadherin陽性の細胞である、請求項1~6のいずれか1項に記載の方法。
- 培地がbFGF、EGF、TGF、NGF、及びWnt3aから選ばれる少なくとも1以上のサイトカインを含む、請求項1~7のいずれか1項に記載の方法。
- 培地が血清代替物を含む、請求項1~8のいずれか1項に記載の方法。
- 培地が、さらにレチノイン酸、βメルカプトエタノール、ピルビン酸ナトリウム、及びアスコルビン酸から選ばれる1又は2以上を含む、請求項8又は9に記載の方法。
- コラーゲン、ラミニン、フィブロネクチン、マトリゲルTM、ポリ−L−オルニチン/ラミニン、又はポリ−D−リジン(PDL)でコーティングした培養容器を用いて接着培養することを特徴とする、請求項1~10のいずれか1項に記載の方法。
- 請求項1~11のいずれか1項記載の方法によって調製された角膜内皮前駆細胞を、角膜内皮細胞に分化誘導することを特徴とする、角膜内皮細胞の調製方法。
- 分化誘導がTGF−β2を含む無血清培地、又は血清を含む培地を用いて行われる、請求項12に記載の方法。
- 請求項1~11のいずれか1項に記載の方法で調製した角膜内皮前駆細胞、あるいは請求項12又は13に記載の方法で調製した角膜内皮細胞を高分子膜上で培養することを特徴とする、角膜内皮細胞シートの作製方法。
- 高分子膜が、コンドロイチン硫酸、デルマタン硫酸、ヒアルロン酸、ヘパラン硫酸、ヘパリン、及びケラタン硫酸を含むグリコサミノグリカン;アテロコラーゲン、アルカリ処理コラーゲン、及びゼラチン;ケラチン;プロテオグリカン;アルギン酸、キトサン、及びヒアルロン酸;ポリアミノ酸(ポリ乳酸);セルロース;(メタ)アクリルアミド化合物、N−(若しくはN,N−ジ)アルキル置換(メタ)アクリルアミド誘導体、ビニルエーテル誘導体、及びこれらの共重合体を含む温度応答性ポリマー;からなる群より選ばれるいずれか1又は2以上を含むものである、請求項14に記載の方法。
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US9347041B2 (en) | 2016-05-24 |
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EP2733201A4 (en) | 2015-01-21 |
US20140170751A1 (en) | 2014-06-19 |
EP2733201A1 (en) | 2014-05-21 |
JPWO2013012087A1 (ja) | 2015-02-23 |
JP5761827B2 (ja) | 2015-08-12 |
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