JP5985207B2 - Method for producing retinal pigment epithelial cells - Google Patents

Description

  The present invention relates to a method for producing retinal pigment epithelial cells and the like.

The retinal pigment epithelial cells are pigment epithelial cells existing outside the neural retina, and play an important role in maintaining photoreceptor cells that receive light. Abnormalities in retinal pigment epithelial cells cause diseases such as macular degeneration, so the use of retinal pigment epithelial cells as a cell therapy transplant material, disease research material, and drug discovery material for these diseases Expected.
It is also considered to be a promising material for toxicity studies of chemical substances, toxicity studies such as phototoxicity, and toxicity tests. Retinal pigment epithelial cells are required for carrying out the above studies and tests. To date, reports of producing retinal pigment epithelial cells using pluripotent stem cells are known (Non-patent Document 1). However, there are no reports of producing retinal pigment epithelial cells with high efficiency.

Maria Idelson, Ruslana Alper, Alexey Obolensky, Etti Ben-Shushan, Itzhak Hemo, Nurit Yachimovich-Cohen, Hanita Khaner, Yoav Smith, Ofer Wiser, Michal Gropp, Malkiel A. Cohen, Sharona Even-Ram, Yael Berman-Zaken, Limor Matzrafi, Gideon Rechavi, Eyal Banin, and Benjamin Reubinoff (2009) Directed Differentiation of HumanEmbryonic Stem Cells into Functional Retinal Pigment Epithelium Cells.Cell Stem Cell 5, 396-408

  Development of a technique for producing retinal pigment epithelial cells with high efficiency has been eagerly desired.

As a result of intensive studies in view of such a situation, the present inventors have reached the present invention.
That is, the present invention
[1] A method for producing retinal pigment epithelial cells, comprising the following steps (1) to (4):
(1) First step of forming pluripotent stem cell aggregates by suspension culture in a serum-free medium containing a Wnt signal pathway inhibitory substance (2) Coagulation formed in the first step Second step of suspension culture of aggregate in serum-free medium containing basement membrane preparation (3) Third step of suspension culture of aggregate cultured in second step in serum medium, and (4) First Aggregates cultured in three steps are treated with a serum-free medium or serum medium containing a Wnt signal pathway agonist (however, the serum-free medium and serum medium may be Sonic hedgehog (hereinafter sometimes referred to as “Shh”)). The fourth step of suspension culture in the signal pathway agent (2) [2] In the fourth step, the aggregate cultured in the third step contains a Wnt signal pathway agonist and an Activin signal pathway agonist. Suspension culture in serum medium or serum medium The [1] The method for producing retinal pigment epithelial cells, wherein a is a step.
[3] The method for producing retinal pigment epithelial cells according to [1] or [2], wherein the pluripotent stem cells are primate pluripotent stem cells.
[4] The method for producing retinal pigment epithelial cells according to any one of [1] to [3], wherein the pluripotent stem cells are human pluripotent stem cells.
[5] The above-mentioned [1] to [4], wherein the basement membrane preparation is at least one extracellular matrix molecule selected from the group consisting of laminin, type IV collagen, heparan sulfate proteoglycan and entactin. A method for producing a retinal pigment epithelial cell according to any one of the above.
[6] The retinal pigment epithelial cells according to any one of [1] to [5], wherein the first to third steps are performed in the presence of a knockout serum substitute (hereinafter sometimes referred to as “KSR”). Manufacturing method.
[7] Use as a reagent for evaluating toxicity / drug efficacy of retinal pigment epithelial cells produced by the production method according to any one of [1] to [6].
[8] Use of a retinal pigment epithelial cell produced by the production method according to any one of [1] to [6] as a biomaterial for transplantation.

  According to the production method of the present invention, retinal pigment epithelial cells can be produced with high efficiency. Therefore, the method of the present invention efficiently produces retinal pigment epithelial cells, which are reagents for use in research, testing and treatment, for the purpose of evaluating toxicity and drug efficacy of chemical substances and the like, transplantation materials for cell therapy, etc. It is very useful from the viewpoint of doing.

FIG. 1 shows that aggregates are formed by suspension culture in a serum-free medium containing a Wnt signal pathway inhibitor, and the formed aggregates are suspended and cultured in the presence of a basement membrane preparation in a serum-free medium. It is a figure which shows the bright field of the aggregate manufactured by culture | cultivating the obtained aggregate in the culture medium containing a serum, and culture | cultivating the cultured aggregate in the culture medium containing a Wnt signal pathway effect | action substance. Fig. 2 shows the formation of aggregates by suspension culture in a serum-free medium containing a Wnt signal pathway inhibitor, and the formed aggregates are suspended and cultured in the presence of a basement membrane preparation in a serum-free medium. Shows a bright field of an aggregate produced by culturing the aggregate in a medium containing serum and culturing the cultured aggregate in a medium containing a Wnt signal pathway agonist and an ActivinA signal pathway agonist It is.

  Hereinafter, embodiments for carrying out the present invention will be described in detail.

  The “transformant” in the present invention means all or part of a living organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant may be referred to as a transformed cell, a transformed tissue, a transformed host, or the like depending on the subject. The cell used in the present invention may be a transformant.

  Examples of prokaryotic cells used in the genetic manipulation technique in the present invention include, for example, prokaryotic cells belonging to the genus Eschericia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Eschericia XL1- Blue, Eschericia XL2-Blue, Eschericia DH1, etc. can be mentioned. Such cells are specifically described in, for example, “Molecular Cloning (3rd edition)” by Sambrook, J and Russell, DW, Appendix 3 (Volume 3), Vectors and Bacterial strains. A3.2 (Cold Spring Harbor USA 2001). Have been described.

The “vector” in the present invention means a vector capable of transferring a target polynucleotide sequence to a target cell. Examples of such vectors include autonomous replication in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals and individual plants, or integration into chromosomes. Examples thereof include those containing a promoter at a position suitable for polynucleotide transcription.
Among such vectors, a vector suitable for cloning may be referred to as a “cloning vector”. Such cloning vectors usually contain multiple cloning sites containing multiple restriction enzyme sites. At present, there are a large number of vectors that can be used for gene cloning in the technical field, and they are sold by vendors with different names (for example, types and sequences of restriction enzymes at multiple cloning sites). ing. For example, “Molecular Cloning (3rd edition)” by Sambrook, J and Russell, DW, Appendix 3 (Volume 3), Vectors and Bacterial strains. A3.2 (Cold Spring Harbor USA, 2001)) (The seller is also described), and those skilled in the art can appropriately use these according to the purpose.

  The “vector” in the present invention also includes “expression vector”, “reporter vector”, and “recombinant vector”. The “expression vector” means a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. Examples of the “regulatory element” include those containing a terminator, a selection marker such as a drug resistance gene, and an enhancer. It is well known to those skilled in the art that the type of expression vector of an organism (eg, animal) and the type of regulatory element used can vary depending on the host cell.

  In the context of the present invention, “recombinant vectors” include, for example, (a) a lambda FIX vector (phage vector) for screening genomic libraries, and (b) a lambda ZAP for screening cDNA. In order to clone vectors (phage vectors) and (c) genomic DNA, for example, pBluescript II SK +/−, pGEM, pCR2.1 vector (plasmid vector) and the like can be mentioned. Examples of the “expression vector” include pSV2 / neo vector, pcDNA vector, pUC18 vector, pUC19 vector, pRc / RSV vector, pLenti6 / V5-Dest vector, pAd / CMV / V5-DEST vector, pDON-AI-2 / neo vector, pMEI-5 / neo vector, etc. (plasmid vector). Examples of the “reporter vector” include pGL2 vector, pGL3 vector, pGL4.10 vector, pGL4.11 vector, pGL4.12 vector, pGL4.70 vector, pGL4.71 vector, pGL4.72 vector, pSLG vector, pSLO. Examples include vectors, pSLR vectors, pEGFP vectors, pAcGFP vectors, pDsRed vectors, and the like. Such a vector may be appropriately used with reference to the aforementioned Molecular Cloning magazine.

  In the context of the present invention, examples of techniques for introducing nucleic acid molecules into cells include transformation, transduction, transfection, and the like. Specific examples of such introduction techniques include Ausubel FA et al. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J. et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd. Examples include Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method”, Yodosha, 1997, and the like. Examples of the technique for confirming that the gene has been introduced into the cell include Northern blot analysis, Western blot analysis, and other well-known conventional techniques.

  In connection with the present invention, vector introduction methods include, for example, transfection, transduction, transformation and the like (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, particle gun (gene gun ) And the like.

  In the present invention, a “stem cell” is a cell that maintains the same differentiation ability even after cell division, and can be regenerated when the tissue is damaged. Here, the stem cells are embryonic stem cells (ES cells) or tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells or somatic stem cells) or induced pluripotent stem cells (iPS cells: induced pluripotent stem cell cells). But you are not limited to them. It is known that the above stem cell-derived tissue cells can differentiate normal cells close to a living body, as can be seen from the fact that tissue regeneration is possible.

  Stem cells can be obtained from a predetermined institution, and commercially available products can also be purchased. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute of Regenerative Medicine, Kyoto University. EB5 cells, which are mouse embryonic stem cells, can be obtained from RIKEN, and the D3 strain can be obtained from ATCC.

  Stem cells can be maintained and cultured by a method known per se. For example, stem cells can be maintained by culture with feeder-free cells supplemented with fetal calf serum (FCS), Knockout Serum Replacement (KSR), LIF.

  The “pluripotent stem cell” in the present invention can be cultured in vitro and all cells constituting the living body excluding the placenta (tissues derived from the three germ layers (ectoderm, mesoderm, endoderm)) Stem cells having the ability to differentiate into (pluripotency), including embryonic stem cells (ES cells). A “pluripotent stem cell” is obtained from a fertilized egg, a cloned embryo, a germ stem cell, or a stem cell in tissue. Also included are cells that are artificially provided with differentiating pluripotency similar to embryonic stem cells by introducing several types of genes into somatic cells (also called artificial pluripotent stem cells). Pluripotent stem cells can be prepared by a method known per se. For example, Cell 131 (5) pp. 861-872, Cell 126 (4) pp. The method of 663-676 is mentioned.

  The “embryonic stem cell (ES cell)” in the present invention is a stem cell having self-renewal ability and pluripotency (ie, pluripotency “pluripotency”), and a pluripotent stem cell derived from an early embryo Say. Embryonic stem cells were first established in 1981, and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and are being used in regenerative medicine.

  The “artificial pluripotent stem cell” in the present invention is a cell in which differentiated cells such as fibroblasts are directly initialized by expression of several types of genes such as Oct3 / 4, Sox2, Klf4, Myc, etc. to induce pluripotency. In 2006, it was established with mouse cells by Yamanaka et al. (Takahashi K, Yamanaka S. Cell. 2006, 126 (4), p663-676). Established in human fibroblasts in 2007, it has pluripotency similar to embryonic stem cells (Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Cell. 2007, 131 ( 5), p861-872.Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA., Science 2007, 318 (5858), p1917-1920., Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Nat Biotechnol., 2008, 26 (1), p101-106).

  The “retinal pigment epithelial cell” in the present invention means an epithelial cell existing outside the neural retinal tissue in the living retina. If it is those skilled in the art, whether it is a retinal pigment epithelial cell, for example, expression of a cell marker (RPE65 (pigment epithelial cell), Mitf (pigment epithelial cell) etc.), presence of melanin granule, characteristic cell of polygon It can be easily confirmed by the form.

  The medium used in the present invention can be prepared using a medium used for culturing animal cells as a basal medium. As the basal medium, for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham medium, RPMI 1640 medium , Fischer's medium, and mixed media thereof are not particularly limited as long as they can be used for animal cell culture.

  The “serum-free medium” in the present invention means a medium that does not contain unconditioned or unpurified serum. A medium mixed with purified blood-derived components or animal tissue-derived components (for example, growth factors) corresponds to a serum-free medium unless it contains unadjusted or unpurified serum.

  The medium is not particularly limited as long as it is as described above. However, from the viewpoint of avoiding complicated preparation, a serum-free medium (GMEM or DMEM, 0.1 mM 2-mercaptoethanol) to which an appropriate amount (for example, 1-20%) of commercially available KSR is added as such a serum-free medium. 0.1 mM non-essential amino acids Mix, 1 mM sodium pyruvate).

  The serum-free medium may contain a serum substitute. The serum substitute may appropriately contain, for example, albumin, transferrin, fatty acid, collagen precursor, trace element, 2-mercaptoethanol or 3'thiolglycerol, or an equivalent thereof. Such serum replacement can be prepared, for example, by the method described in WO98 / 30679. Moreover, in order to implement the method of this invention more simply, a serum substitute can utilize. Examples of such commercially available serum substitutes include Chemically-defined Lipid concentrated (Gibco) and Glutamax (Gibco).

  The serum-free medium used in suspension culture contains fatty acids or lipids, amino acids (for example, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like. Can be contained.

  The “serum medium” in the present invention means a medium containing unadjusted or unpurified serum. The medium is not particularly limited as long as it is as described above. Moreover, fatty acids or lipids, amino acids (for example, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like can be contained.

In the present invention, “suspension culture” refers to culturing pluripotent stem cells that have formed aggregates formed in the first step in a culture medium under non-adhesive conditions with respect to a cell culture vessel. Say.

  The incubator used in suspension culture is not particularly limited as long as the suspension culture of cells is possible. Examples of such an incubator include flasks, flasks for tissue culture, dishes, petri dishes, and tissue cultures. A dish, a multi-dish, a microplate, a microwell plate, a micropore, a multiplate, a multiwell plate, a chamber slide, a petri dish, a tube, a tray, a culture bag, and a roller bottle can be mentioned. These incubators are preferably non-cell-adhesive. As the non-cell-adhesive incubator, those in which the surface of the incubator has not been artificially treated (for example, coating treatment with an extracellular matrix or the like) for the purpose of improving adhesion to cells can be used.

The present invention is a method for producing retinal pigment epithelial cells, comprising the following steps (1) to (4).
(1) First step of forming pluripotent stem cell aggregates by suspension culture in a serum-free medium containing a Wnt signal pathway inhibitory substance (2) Coagulation formed in the first step The second step of culturing the aggregate in a serum-free medium containing a basement membrane preparation (3) The third step of culturing the aggregate cultured in the second step in a serum medium, and (4) the third The fourth step of culturing the aggregate cultured in the step in a serum-free medium or serum medium containing a Wnt signal pathway agent (but not containing a sonic hedgehog signal pathway agent)

(1) 1st process The 1st process of forming the pluripotent stem cell aggregate in the serum-free medium containing a Wnt signal pathway inhibitory substance is demonstrated.

  The Wnt signal pathway inhibitor is not particularly limited as long as it can suppress signal transduction mediated by Wnt. Examples of the Wnt signal pathway inhibitor include Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, CKI-7 (N- (2- Aminoethyl) -5-chloro-isoquinoline-8-sulfonamide), D4476 (4- {4- (2,3-dihydrobenzo [1,4] dioxin-6-yl) -5-pyridin-2-yl- 1H-imidazol-2-yl} benzamide), IWR-1-endo (IWR1e), IWP-2 and the like.

  The concentration of the Wnt signal pathway inhibitor used in the production method of the present invention may be any concentration that forms an aggregate of pluripotent stem cells. For example, in the case of a normal Wnt signal pathway inhibitor such as IWR1e, it is added at a concentration of about 0.1 μM to 100 μM, preferably about 1 μM to 10 μM, more preferably around 3 μM.

  The Wnt signal pathway inhibitor may be added to the serum-free medium before suspension culture, or may be added to the medium within a few days (for example, within 5 days) after the start of suspension culture. Preferably, the Wnt signal pathway inhibitor is added to the serum-free medium within 5 days after the start of suspension culture, more preferably within 3 days, and most preferably simultaneously with the start of suspension culture. In addition, the cells are cultured until the 18th day, more preferably the 12th day after the start of suspension culture, with the addition of a Wnt signal pathway inhibitor.

Culture conditions such as culture temperature and CO ₂ concentration in the first step can be set as appropriate. The culture temperature is not particularly limited, but is, for example, about 30 to 40 ° C., preferably about 37 ° C. The CO ₂ concentration is, for example, about 1 to 10%, preferably about 5%.

The “aggregate” in the present invention refers to a mass formed by aggregation of cells dispersed in the medium. In the “aggregate” in the present invention, cells dispersed at the start of suspension culture are formed. And aggregates that have already been formed at the start of suspension culture.
“Aggregate formation” means that when a cell is aggregated to form an aggregate of cells and cultured, a qualitatively uniform cell is formed by “aggregating a certain number of dispersed stem cells rapidly”. It means forming an aggregate.

  Experimental operations for forming aggregates include, for example, a method of confining cells in a small space using a plate with a small well (96-well plate) or a micropore, or a short centrifugation using a small centrifuge tube. Examples thereof include a method for aggregating cells.

  The concentration of pluripotent stem cells in the first step can be appropriately set by those skilled in the art so that aggregates of pluripotent stem cells can be formed more uniformly and efficiently. The concentration of pluripotent stem cells at the time of aggregate formation is not particularly limited as long as it is a concentration capable of forming a uniform aggregate of stem cells. For example, when suspension culture of human ES cells using a 96-well microwell plate, About 1 × 10 3 to about 5 × 10 4 cells, preferably about 3 × 10 3 to about 3 × 10 4 cells, more preferably about 5 × 10 3 to a well A solution prepared so as to be about 2 × 10 4 cells, most preferably about 9 × 10 3 cells, is added, and the plate is allowed to stand to form aggregates.

  The suspension culture time required to form the aggregate can be determined as appropriate depending on the pluripotent stem cell used as long as the cells can be rapidly aggregated, but can be formed to form a uniform aggregate. It is desirable to be as short as possible. For example, in the case of human ES cells, it is desirable to form aggregates preferably within 24 hours, more preferably within 12 hours. A person skilled in the art can appropriately adjust the time until the formation of the aggregates by adjusting a tool for aggregating cells, centrifugation conditions, and the like.

  The formation of aggregates of pluripotent stem cells is due to the size and number of the aggregates, the macroscopic morphology, the microscopic morphology and its homogeneity by tissue staining analysis, the expression and the homogeneity of differentiation and undifferentiation markers Those skilled in the art can judge based on the expression control and differentiation of differentiation markers, the reproducibility between aggregates of differentiation efficiency, and the like.

(2) Second Step A second step of suspension culture of the aggregate formed in the first step in a serum-free medium containing a basement membrane preparation will be described.

  “Basement membrane preparation” controls epithelial cell-like cell morphology, differentiation, proliferation, motility, functional expression, etc. when desired cells with basement membrane-forming ability are seeded and cultured on it It includes a basement membrane component that has a function. Here, the “basement membrane component” refers to a thin membrane-like extracellular matrix molecule that exists between an epithelial cell layer and a stromal cell layer in an animal tissue. Basement membrane preparation is prepared by removing cells with basement membrane-forming ability that adhere to the support through the basement membrane using a solution or alkaline solution that has lipid-dissolving ability of the cells. can do. Preferred basement membrane preparations include commercially available basement membrane components (eg, Matrigel) and known extracellular matrix molecules (eg, laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.) as basement membrane components. Things.

  Matrigel is a basement membrane preparation from Engelbreth Holm Swarn (EHS) mouse sarcoma. The main components of Matrigel are type IV collagen, laminin, heparan sulfate proteoglycan and entactin, but in addition to these, TGF-β, fibroblast growth factor (FGF), tissue plasminogen activator, and EHS tumor are naturally produced Growth factors are included. Matrigel's “growth factor reduced product” has a lower concentration of growth factors than normal Matrigel, with standard concentrations of <0.5 ng / ml for EGF, <0.2 ng / ml for NGF, and <5 pg for PDGF. / Ml, IGF-1 is 5 ng / ml, and TGF-β is 1.7 ng / ml. In the method of the present invention, it is preferable to use “growth factor reduced products”.

  The concentration of the basement membrane preparation added to the serum-free medium in the suspension culture in the second step is not particularly limited as long as the epithelial structure of nerve tissue (for example, retinal tissue) is stably maintained. For example, when using Martigel Preferably includes a volume of 1/20 to 1/200 of the culture solution, more preferably a volume of about 1/100. The basement membrane preparation may be already added to the medium at the start of stem cell culture, but is preferably added to the serum-free medium within 5 days after the start of suspension culture, more preferably within 2 days of the start of suspension culture.

The serum-free medium used in the second step can be the same as the serum-free medium used in the first step, or can be replaced with a new serum-free medium.
When the serum-free medium used in the first step is used in this step as it is, a “basement membrane preparation” may be added to the medium.

  The serum-free medium used for suspension culture in the first step and the second step is not particularly limited as long as it is as described above. However, from the viewpoint of avoiding complicated preparation, a serum-free medium (GMEM or dMEM, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential) to which an appropriate amount of commercially available KSR (Knockout Serum Replacement) is added as such a serum-free medium The amino acids Mix, 1 mM sodium pyruvate) are preferably used. The dose of KSR to the serum-free medium is not particularly limited. For example, in the case of human ES cells, it is usually 1 to 20%, preferably 2 to 20%.

Culture conditions such as culture temperature and CO ₂ concentration in the second step can be set as appropriate. The culture temperature is not particularly limited, but is, for example, about 30 to 40 ° C., preferably about 37 ° C. The CO ₂ concentration is, for example, about 1 to 10%, preferably about 5%.

(3) Third step The third step of suspension culture of the aggregate cultured in the second step in a serum medium will be described.

  As the serum medium used in the third step, one obtained by directly adding serum to the serum-free medium used for culturing in the second step may be used, or one replaced with a new serum medium may be used.

Examples of the serum added to the medium in this step include bovine serum, calf serum, fetal calf serum, horse serum, foal serum, fetal bovine serum, and rabbit serum. Sera from mammals such as baby rabbit serum, rabbit fetal serum, and human serum can be used.

  For example, when human ES cells are suspended, serum is added on or after the 7th day, more preferably on or after the 9th day, and most preferably on the 12th day after the start of suspension culture. Regarding the serum concentration, it is added at about 1 to 30%, preferably about 3 to 20%, more preferably around 10%.

The serum medium used in the third step is not particularly limited as long as it is as described above, but the serum-free medium (GMEM or DMEM, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid Mix, 1 mM pyruvin). It is preferable to use a solution obtained by adding serum to (sodium acid).
Moreover, you may use what added a suitable quantity of commercially available KSR (Knockout Serum Replacement) as this serum culture medium.

  In the third step, the production efficiency of retinal pigment epithelial cells can be increased by adding an Shh signal pathway agonist in addition to serum.

  The substance acting on the Shh signal pathway is not particularly limited as long as it can enhance signal transduction mediated by Shh. Examples of substances acting on the Shh signal pathway include proteins belonging to the Hedgehog family (for example, Shh), Shh receptors, Shh receptor agonists, Purmorphamine, SAG and the like.

  The concentration of the Shh signal pathway agonist used in the production method of the present invention is about 0.1 nM to 10 μM, preferably about 10 nM to 1 μM, more preferably about 100 nM in the case of a normal Shh signal pathway agonist such as SAG. Add in concentration.

(4) Fourth step Aggregates cultured in the third step are cultured in suspension in a serum-free medium or serum medium containing a Wnt signal pathway agent (but not containing a sonic hedgehog signal pathway agent). The fourth step will be described.

  Examples of Wnt signal pathway agonists include proteins belonging to the Wnt family, Wnt receptors, Wnt receptor agonists, GSK3β inhibitors (for example, 6-Bromoindirubin-3′-oxime (BIO), CHIR99021, Kenpaullone) and the like. It is done.

  The concentration of the Wnt signal pathway agonist used in this method is about 0.1 μM to 100 μM, preferably about 1 μM to 30 μM, more preferably around 3 μM in the case of a normal Wnt signal pathway agonist such as CHIR99021. Add in.

  For example, when human ES cells are used, the Wnt signal pathway agonist is added after the 12th day after the start of production, and most preferably on the 15th day. At this time, a medium not containing the Wnt signal pathway inhibitor added in the first step and the Shh signal pathway agonist added in the third step is used.

  In the fourth step, it is preferably cultured in a serum-free medium or serum medium containing a Wnt signal pathway agonist and an Activin signal pathway agonist.

  The Activin signal pathway agonist is not particularly limited as long as it can enhance signal transduction mediated by Activin. Examples of the Activin signal pathway agonist include proteins belonging to the Activin family (for example, Activin A, Activin B, Activin CActivin AB, etc.), Activin receptors, Activin receptor agonists, and the like.

  The concentration of the Activin signal pathway agonist used in the fourth step is, for example, 1 ng / ml to 10 ug in the case of a normal Activin signal pathway agonist such as Recombinant Human / Mouse / Rat Activin A (R & D systems # 338-AC). / ml, preferably about 10 ng / ml to 1 ug / ml, more preferably around 100 ng / ml.

Since the retinal pigment epithelial cells thus produced are present on the surface of the aggregate, they can be easily confirmed by microscopic observation or the like. It is also possible to obtain high-purity retinal pigment epithelial cells by using, for example, dispersion treatment (for example, trypsin / EDTA treatment) using an aggregate in which retinal pigment epithelial cells are present, and sorting using FACS. It is also possible to physically cut out retinal pigment epithelial cells from the aggregates using tweezers or the like and culture them. The dispersed or excised retinal pigment epithelial cells can be cultured under adhesive conditions. In the case of adhesion culture, it is preferable to use a cell adhesion incubator, for example, an incubator coated with an extracellular matrix or the like (for example, poly-D-lysine, laminin, fibronectin). In addition, culture conditions such as culture temperature, CO ₂ concentration, and O ₂ concentration in adhesion culture can be easily determined by those skilled in the art. In this case, the culture may be performed in the presence of serum, a known growth factor, an additive for promoting growth, or a chemical substance. Examples of known growth factors include EGF and FGF. Examples of additives that promote growth include N2 supplement (Invitrogen) and B27 supplement (Invitrogen).

(5) Use of Retinal Pigment Epithelial Cells as Reagents for Toxicity and Drug Efficacy Retinal pigment epithelial cells produced by the production method of the present invention have characteristics very similar to those of living retinal pigment epithelial cells. It can be used as a screening for therapeutic drugs for diseases based on cell damage, as a cell therapy transplant material, as a disease research material, or as a drug discovery material for therapeutic drugs in cell damage caused by other causes. It can also be used for toxicity studies such as phototoxicity, toxicity tests, etc. in the evaluation of toxicity and drug efficacy of chemical substances.

(6) Use of retinal pigment epithelial cells as a biomaterial for transplantation The retinal pigment epithelial cells produced by the production method of the present invention replenish the cells and the damaged tissue itself in a cell damage state (for example, It can be used as a biomaterial for transplantation used for, for example, transplantation surgery.

The present invention will be described more specifically with reference to the following examples. However, the examples are merely illustrative of the present invention and do not limit the scope of the present invention.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1: Production of highly efficient retinal pigment epithelial cells using human ES cells (Method)
Human ES cells (KhES-1) were cultured according to the method described in “Ueno, M. et al. PNAS 2006” and “Watanabe, K. et al. Nat Biotech 2007” and used for experiments. The medium used was a DMEM / F12 medium (Invitrogen) supplemented with 20% KSR (Knockout Serum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvic acid, 5-10 ng / ml bFGF. For the formation of retinal tissue by suspension culture, ES cells are monodispersed using 0.25% trypsin-EDTA (Invitrogen), and a non-cell-adhesive 96-well culture plate (Sumilon Spheroid Plate, Sumitomo Bakelite Co., Ltd.) The suspension was suspended in 100 μl of a serum-free medium so that 9 × 10 3 cells per well were formed, and an aggregate was rapidly formed, followed by culturing at 37 ° C. and 5% CO ₂ . For the serum-free medium, use a serum-free medium with 20% KSR, 0.1 mM 2-mercaptoethanol, 1 mM pyruvic acid, 20 μM Y27632, Wnt signal pathway inhibitor (3 μM IWR1e) added to G-MEM medium. It was. In addition, from the second day of suspension culture, 1/100 amount of Matrigel per volume was added and suspension culture was performed. On the 12th day from the start of suspension culture, the aggregate was transferred to a serum-free medium containing no Wnt signal pathway inhibitor, and 1/10 amount of fetal bovine serum per volume was added and cultured. From the 15th day, suspension culture was carried out in a medium containing a Wnt signal pathway agonist (3 μM CHIR99021).
(result)
When produced by the above method, a population of retinal pigment epithelial cells that appeared black on the surface of almost all aggregates appeared (FIG. 1).

Example 2: Production of retinal pigment epithelial cells using human ES cells (Method)
Human ES cells (KhES-1) were cultured in suspension according to the method described in “Ueno, M. et al. PNAS 2006” and “Watanabe, K. et al. Nat Biotech 2007” and used for experiments. The medium used was a DMEM / F12 medium (Invitrogen) supplemented with 20% KSR (Knockout Serum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvic acid, 5-10 ng / ml bFGF. For the production of retinal tissue by suspension culture, ES cells are monodispersed using 0.25% trypsin-EDTA (Invitrogen), and a non-cell-adhesive 96-well culture plate (Sumilon Spheroid Plate, Sumitomo Bakelite Co., Ltd.) is used. The suspension was suspended in 100 μl of serum-free medium so that 9 × 10 3 cells per well were formed, and an aggregate was rapidly formed, followed by suspension culture at 37 ° C. and 5% CO ₂ . For the serum-free medium, use a serum-free medium with 20% KSR, 0.1 mM 2-mercaptoethanol, 1 mM pyruvic acid, 20 μM Y27632, Wnt signal pathway inhibitor (3 μM IWR1e) added to G-MEM medium. It was. Further, from the second day of culture, 1/100 volume of Matrigel per volume was added, and suspension culture was performed. On the 12th day after the start of suspension culture, the aggregate was transferred to a serum-free medium containing no Wnt signal pathway inhibitor, and cultured with 1/10 volume of fetal bovine serum added per volume. From the 15th day, suspension culture was carried out in a medium containing 100 ng / ml of Wnt signal pathway agonist (3 μM CHIR99021) and Activin signal pathway agonist (Recombinant Human / Mouse / Rat Activin A (R & D systems # 338-AC)).
(result)
When manufactured by the above-described method, retinal pigment epithelial cells exhibiting black color appeared on the surface of almost all aggregates. Furthermore, almost the entire surface of the aggregate was covered with retinal pigment epithelial cells (FIG. 2), and retinal pigment epithelial cells were produced with surprisingly high efficiency.

Example 3: Transplantation of retinal pigment epithelial cells produced from human ES cells into the eye After scleral incision of the eyeball, an injection needle is inserted into the vitreous from the scleral incision to reduce intraocular pressure. An intraocular perfusate is injected from the scleral incision into the subretinal space using a cell transplant needle to artificially form a shallow retinal detachment state. Retinal pigment epithelial cells are transplanted into the formed space using a cell transplant needle or a cell sheet transplant device.

  According to the method of the present invention, retinal pigment epithelial cells can be produced with high efficiency.

Claims (6)

The manufacturing method of the retinal pigment epithelial cell characterized by including the process of following (1)-(4).
(1) First step (2) In the first step, pluripotent stem cells are aggregated by suspension culture in a serum-free medium containing a Wnt signaling pathway inhibitor and a serum substitute. Second step of suspension culture of the formed aggregate in a serum-free medium containing a basement membrane preparation and a serum substitute (3) First step of suspension-aggregating the aggregate cultured in the second step in a serum medium third step, and (4) are aggregates cultured in the third step, GSK3 [beta inhibitor including blood Kiyoshi培地(where the previous Kichi Qing medium free of Sonic hedgehog signaling pathway effects thereof) The fourth step of floating culture in The fourth step, the third step in cultured the aggregate, according to claim 1, characterized in that the step of suspension culture in GSK3β inhibitor and Activin signaling pathway agonist and including serum medium A method for producing retinal pigment epithelial cells. The method for producing retinal pigment epithelial cells according to claim 1 or 2, wherein the pluripotent stem cells are primate pluripotent stem cells. The method for producing retinal pigment epithelial cells according to any one of claims 1 to 3, wherein the pluripotent stem cells are human pluripotent stem cells. The retinal pigment according to any one of claims 1 to 4, wherein the basement membrane preparation is at least one extracellular matrix molecule selected from the group consisting of laminin, type IV collagen, heparan sulfate proteoglycan and entactin. A method for producing epithelial cells. The method for producing retinal pigment epithelial cells according to any one of claims 1 to 5, wherein the first to third steps are performed in the presence of a knockout serum substitute.

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