JP5985208B2 - Method for manufacturing retinal tissue - Google Patents

Description

  The present invention relates to a retinal tissue manufacturing method and the like.

  As a method for producing retinal tissue from pluripotent stem cells, Non-Patent Document 1 and Patent Document 1 form a uniform pluripotent stem cell aggregate in a serum-free medium, and this is the presence of a basement membrane preparation. It is described that retinal tissue can be produced in a test tube by suspension culture below.

International Publication No. 2011/055855

Mototsugu Eiraku, Nozomu Takata, Hiroki Ishibashi, Masako Kawada, Eriko Sakakura, Satoru Okuda, Kiyotoshi Sekiguchi, Taiji Adachi & Yoshiki Sasai (2011) Self-organizing optic-cup morphogenesis in three-dimensional culture.Nature Volume: 472, Pages: 51 -56

  A method for producing retinal tissue with high efficiency has been 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 includes [1] a method for producing retinal tissue, which comprises the following steps (1) to (3).
(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 [2] The method for producing retinal tissue according to [1] above, wherein the sex stem cell is a primate pluripotent stem cell.
[3] The method for producing a retinal tissue according to [1], wherein the pluripotent stem cell is a human pluripotent stem cell.
[4] The above-mentioned [1] to [3], 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 tissue according to any one of the above.
[5] Any one of [1] to [4], wherein the first to third steps are performed in the presence of a knockout serum substitute (hereinafter sometimes referred to as “KSR”). Manufacturing method of retinal tissue.
[6] Use as a reagent for evaluating toxicity / drug efficacy of retinal tissue produced by the production method according to any one of [1] to [5].
[7] Use of a retinal tissue produced by the production method according to any one of [1] to [5] as a biomaterial for transplantation.

  According to the manufacturing method of the present invention, it is possible to manufacture retinal tissue with high efficiency. Furthermore, the production method of the present invention efficiently produces a cell group (photocell, optic nerve, etc.) constituting a retinal tissue and has a tissue structure for the purpose of toxicity and drug efficacy evaluation of a chemical substance, cell therapy, and the like. For the purpose of evaluating toxicity and drug efficacy using retinal tissue and applying it to transplantation materials for retinal tissue transplantation treatment, it is possible to efficiently produce retinal tissue as a “reagent” for use in testing and treatment.

Figure 1 shows a bright-field image of human pluripotent stem cell-derived aggregates on the 25th day from the start of suspension culture, which was prepared by adding only Matrigel (hereinafter sometimes referred to as Matrigel) without adding a Wnt signaling pathway inhibitor. (A) and fluorescence image (B), bright field image (C) and fluorescence image of the start of suspension culture of human pluripotent stem cell-derived aggregates produced by adding a Nodal signal pathway inhibitor and Matrigel (C) and fluorescence image ( D) shows a bright field image (E) and a fluorescence image (F) on the 25th day of suspension culture of human pluripotent stem cell-derived aggregates produced by adding a Wnt signal pathway inhibitor and Matrigel. . Fig. 2 shows the suspension culture of human pluripotent stem cells added with a Wnt signaling pathway inhibitor, followed by suspension culture in the presence of Matrigel, and suspension culture in the absence of fetal calf serum. After culturing human pluripotent stem cells in suspension by adding a bright-field image (A) and fluorescence image (B) of the eye, and a Wnt signaling pathway inhibitor, in the presence of Matrigel, and in the presence of fetal bovine serum After the suspension culture of human pluripotent stem cells by adding a bright field image (C) and fluorescence image (D), Wnt signal pathway inhibitor on the 18th day of suspension culture of suspended aggregates, and in the presence of Matrigel Bright field image (E) on the 18th day of suspension culture after suspension culture and suspension culture in the presence of fetal bovine serum and sonic hedgehog (hereinafter sometimes referred to as “Shh”) signaling pathway agent FIG. 6 is a diagram showing a fluorescence image (F). Figure 3 shows the suspension of human pluripotent stem cells added with a Wnt signaling pathway inhibitor, suspended in the presence of Matrigel, and then suspended in the absence of fetal calf serum. FACS histogram (A) of GFP-expressing cells that make up the aggregate, human pluripotent stem cells added to Wnt signaling pathway inhibitor, suspended in the presence of Matrigel, then suspended in the presence of fetal bovine serum FACS histogram (B) of GFP-expressing cells constituting aggregates on the 18th day from the start of cultured suspension culture. Addition of Wnt signal pathway inhibitor and suspension culture of human pluripotent stem cells, followed by suspension culture in the presence of Matrigel Furthermore, the GFP constituting the aggregate on the 18th day of suspension culture in suspension culture in the presence of fetal bovine serum and Shh signal pathway agonists is the FACS histogram (C) of the expression cells and the GFP strong positive retinal progenitor cells. It is a graph of the percentage of cells (D). FIG. 4 shows the retinal tissues (A, B, C) on the 60th day from the start of the floating culture and the retinal tissues (D, E, F, G, H, I) on the 126th day from the start of the floating culture. It is a figure which shows an immuno-staining result.

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

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.

  The “recombinant vector” in the present invention includes, for example, (a) a lambda FIX vector (phage vector) for screening a genomic library, and (b) a lambda ZAP vector (phage for screening cDNA). For example, pBluescript II SK +/−, pGEM, pCR2.1 vector (plasmid vector) and the like can be used for cloning genomic DNA. 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.

  Examples of techniques for introducing a nucleic acid molecule in the present invention 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.

  The “stem cell” in the present invention is a cell that maintains the same differentiation ability even after cell division, and can regenerate the tissue 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, are available from RIKEN, and the D3 strain is available 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), or 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 “tissue” in the present invention refers to a structure of a cell population having a structure in which a plurality of types of cells having different shapes and properties are three-dimensionally arranged in a certain pattern.

  The “retinal tissue” in the present invention is a layer of at least a plurality of types of cells such as photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal node cells, or progenitor cells constituting each retinal layer in a living retina. It means retinal tissue arranged in three dimensions. Which cell constitutes which retinal layer is determined by a known method such as cell marker (Chx10 (bipolar cell), L7 (bipolar cell), Tuj1 (node cell), Brn3 (node cell), Calretinin) (Amacrine cells), Calbindin (horizontal cells), Recoverin (photocells), Rhodopsin (photocells), RPE65 (pigment epithelial cells), Mitf (pigment epithelial cells) and the like.

  Genetically modified pluripotent stem cells can be prepared, for example, by using homologous recombination techniques. Examples of the chromosomal gene that is modified when the modified pluripotent stem cell is prepared include a histocompatibility antigen gene, a disease-related gene based on a neuronal cell disorder, and the like. Modification of the target gene on the chromosome is the following: Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press (1994); Gene Targeting, A Practicing. Gene targeting, production of mutant mice using ES cells, methods described in Yodosha (1995) and the like can be used.

  Specifically, for example, for isolating a genomic gene of a target gene to be modified (for example, a histocompatibility antigen gene or a disease-related gene), and homologous recombination of the target gene using the isolated genomic gene Create a target vector. By introducing the prepared target vector into a stem cell and selecting a cell that has undergone homologous recombination between the target gene and the target vector, a stem cell with a modified gene on the chromosome can be prepared.

  As a method for isolating a genomic gene of a target gene, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular 7 in BioJ. Known methods. Further, the genomic gene of the target gene can be isolated by using a genomic DNA library screening system (manufactured by Genome Systems), Universal Genome Walker Kits (manufactured by CLONTECH), or the like.

  Generation of target vectors for homologous recombination of target genes and efficient selection of homologous recombinants are described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Biomanual Series 8 Gene Targeting, A mutant mouse using ES cells can be prepared according to the method described in Yodosha (1995). The target vector can be either a replacement type or an insertion type, and as a selection method, methods such as positive selection, promoter selection, negative selection, and poly A selection can be used.

  Examples of a method for selecting a target homologous recombinant from the selected cell lines include Southern hybridization method and PCR method for genomic DNA.

  According to the present invention, retinal tissue can be obtained from human pluripotent stem cells with high efficiency. In addition, since the retinal tissue prepared by the method of the present invention includes neurons specific to each of the retinal layers, photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, or precursors thereof. It is also possible to obtain cells that constitute retinal tissue such as cells. Which cell is obtained from the retinal tissue obtained by the method of the present invention can be confirmed by a method known per se, for example, expression of a cell marker.

  As retinal cell markers, Rax (progenitor cell of retinal), PAX6 (progenitor cell), nestin (expressed in progenitor cell of hypothalamic neuron but not expressed in retinal progenitor cell), Sox1 (expressed in hypothalamic neuroepithelium) , Not expressed in the retina), Crx (progenitor cell of photoreceptor), and the like. In particular, as markers for the retinal layer-specific neurons, Chx10 (bipolar cells), L7 (bipolar cells), Tuj1 (node cells), Brn3 (node cells), Calretinin (amacrine cells), Calbindin (horizontal cells), Examples include, but are not limited to, Rhodopsin (photocell), Recoverin (photocell), RPE65 (pigment epithelial cell), Mitf (pigment epithelial cell) Nrl (rod cell), Rxr-gamma (cone cell).

  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.

“Floating culture” in the present invention means culturing under conditions that do not allow cells or cell masses to adhere to culture equipment or the like.

  The incubator used for the suspension culture is not particularly limited as long as it can perform “suspension culture”, and can be appropriately determined by those skilled in the art. Examples of such incubators include flasks, tissue culture flasks, dishes, petri dishes, tissue culture dishes, multi dishes, micro plates, micro well plates, micro pores, multi plates, multi well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. These incubators are preferably non-cell-adhesive from the viewpoint of suspension culture. 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 “primate” in the present invention refers to a mammal animal belonging to the primate, and examples of the primate include apes of primates such as lemurs, loris, and tsubai, and apes of primates such as monkeys, apes and humans.

The present invention is a method for producing retinal tissue, comprising the following steps (1) to (3).
(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 A second step of suspension culture of the aggregate in a serum-free medium containing a basement membrane preparation; and (3) a third step of suspension culture of the aggregate cultured in the second step in a serum medium.

(1) 1st process The 1st process of forming the aggregate of a pluripotent stem cell by carrying out suspension culture | cultivation of the pluripotent stem cell in the serum-free medium containing a Wnt signal pathway inhibitor 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 the start of suspension culture, or may be added to the serum-free 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 suspension culture is performed until 18 days, more preferably 12 days 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 qualitatively uniform cells by rapidly aggregating a certain number of dispersed stem cells when cells are aggregated to form cell aggregates and suspended in culture. The formation of an aggregate of

  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 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 products that are commercially available as basement membrane components (eg, Matrigel (hereinafter sometimes referred to as Matrigel)), and extracellular matrix molecules known as basement membrane components (eg, laminin, type IV collagen, Heparan sulfate proteoglycan, entactin and the like).

  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 already be 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 after 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 a 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 serum added to the medium in the third 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.

  The serum is added on the 7th day after the start of suspension culture, more preferably on the 9th day and most preferably on the 12th day. 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 tissue can be increased by adding an Shh signal pathway agent 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 this production method is about 0.1 nM to 10 μM, preferably about 10 nM to 1 μM, more preferably around 100 nM in the case of a normal Shh signal pathway agonist such as SAG. Add in.

The retinal tissue thus produced exists so as to cover the surface of the aggregate. Confirmation that the retinal tissue was produced by the production method of the present invention can be confirmed by the immunostaining method described in (4) below.
It is also possible to physically cut out the retinal tissue present on the surface of the aggregate from the aggregate using tweezers or the like. In this case, since a nerve tissue other than the retinal tissue may be formed on the surface of each aggregate, a part of the nerve tissue cut out from the aggregate is cut out and used to immunostain according to (4) By confirming by a method or the like, it is possible to confirm that the tissue is a retinal tissue.

(4) Method for confirming retinal tissue A retinal tissue can be produced through the first to third steps described above. Furthermore, it can be confirmed that the retinal tissue is produced by the first to third steps by the following method.

Aggregates cultured in the third step are suspended in serum medium. Examples of the incubator used in the suspension culture include those described above. Other culture conditions such as culture temperature, CO ₂ concentration, and O ₂ concentration in suspension culture can be set as appropriate. The culture temperature is not particularly limited and 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%. On the other hand, the O ₂ concentration is, for example, 20 to 70%, preferably 20 to 60%, and more preferably 30 to 50%.
The culture time in this step is not particularly limited, but is usually 48 hours or longer, preferably 7 days or longer.

  Confirmation of retinal tissue can be done by fixing the aggregate with a fixative such as paraformaldehyde solution after completion of suspension culture, preparing a frozen section, and confirming that the layer structure is formed by immunostaining. Good. Since retinal progenitor cells (visual cells, horizontal cells, bipolar cells, amacrine cells, retinal node cells) that make up each layer are different from each other, retinal tissue uses antibodies against the above markers expressed in these cells, It can be confirmed that a layer structure is formed by immunostaining.

(5) Use as a reagent for evaluating toxicity / drug efficacy of retinal tissue The retinal tissue produced by the production method of the present invention is used for screening for a therapeutic drug for a disease based on a disorder of the retinal tissue, or a therapeutic drug for cell damage caused by other causes. It can be used as a cell therapy transplant material, disease research material, and drug discovery material. 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.
Examples of diseases based on retinal tissue disorders include organic mercury poisoning, chloroquine retinopathy, retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, and neonatal retinopathy.

(6) Use of retinal tissue as a biomaterial for transplantation The retinal tissue produced by the production method of the present invention replenishes the cells or the damaged tissue itself in a cell damage state (for example, used for transplantation surgery). ) Can be used as a biomaterial for transplantation used for the purpose.

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

(Establishment of RAX knock-in human ES cells)
A human ES cell line in which GFP was knocked in at the RAX locus, which is one of the marker genes of retinal progenitor cells, was prepared.
Zinc Finger Nuclease (ZFN) that specifically cleaves the RAX gene on the genomic DNA of a human ES cell line (KhES-1: human ES cell line established by Kyoto University) was purchased from SigmaAldrich. Neomycin treated with mitomycin C by co-introducing ZFN-encoding mRNA, GFP, and a knock-in vector loaded with a neomycin resistance gene, which is a drug selection gene, by electroporation using single humanized ES cells Seeded onto resistant mouse fibroblasts. From the day after sowing, G418 was added to the medium to select the drug. The obtained colonies of resistant clones were picked up and cultured, and knock-in cells were selected by PCR or Southern blotting to establish a RAX :: GFP knock-in human ES cell line.

Comparative Example 1: Production of retinal tissue using human ES cells (Matrigel addition conditions)
RAX :: GFP knock-in human ES cells (derived from KhES-1) were cultured according to the methods described in “Ueno, M. et al. PNAS 2006” and “Watanabe, K. et al. Nat Biotech 2007” and were used for experiments. Using. 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. ES cells were monodispersed using 0.25% trypsin-EDTA (Invitrogen), and 9 × 10 ^ 3 cells per well of a non-cell-adhesive 96-well culture plate (Sumilon Spheroid Plate, Sumitomo Bakelite) The suspension culture was carried out in 100 μl of serum-free medium at 37 ° C. and 5% CO ₂ . As the serum-free medium at that time, a serum-free medium in which 20% KSR, 0.1 mM 2-mercaptoethanol, 1 mM pyruvic acid and 20 μM Y27632 were added to G-MEM medium was used. In addition, from the second day of suspension culture, 1/100 amount of Matrigel per volume was added and suspension culture was performed. Thereafter, observation with a fluorescence microscope was periodically carried out while continuing floating culture.
As a result, when observation under a fluorescence microscope was performed until the 25th day from the start of suspension culture, some GFP-expressing cells showing that retinal progenitor cells were induced were observed (FIGS. 1A and 1B).

Comparative Example 2: Production of retinal tissue using human ES cells (Nodal signal pathway inhibitor and Matrigel addition conditions)
RAX :: GFP knock-in human ES cells (derived from KhES-1) were cultured according to the methods described in “Ueno, M. et al. PNAS 2006” and “Watanabe, K. et al. Nat Biotech 2007” and were used for experiments. Using. 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. Suspension culture was performed at 37 ° C. and 5% CO ₂ in 100 μl of serum-free medium so that 9 × 10 3 cells per well were obtained. As the serum-free medium, a serum-free medium supplemented with 20% KSR, 0.1 mM 2-mercaptoethanol, 1 mM pyruvic acid, 20 μM Y27632, Nodal signal pathway inhibitor (10 μM SB431542) is used for the 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. Thereafter, suspension culture was continued, and on the 18th day from the start of suspension culture, observation of the fluorescence microscope and confirmation of the ratio of GFP-expressing cells using FACS were performed.
As a result, some GFP-expressing cells were observed (FIGS. 1C and D).

Comparative Example 3: Production of retinal tissue using human ES cells (Wnt signal pathway inhibitor and Matrigel addition conditions)
RAX :: GFP knock-in human ES cells (derived from KhES-1) were cultured according to the methods described in “Ueno, M. et al. PNAS 2006” and “Watanabe, K. et al. Nat Biotech 2007” and were used for experiments. Using. 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. Suspension culture was performed at 37 ° C. and 5% CO ₂ in 100 μl of serum-free medium so that 9 × 10 3 cells per well were obtained. 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. Thereafter, suspension culture was continued, and on the 18th day from the start of suspension culture, observation of the fluorescence microscope and confirmation of the ratio of GFP-expressing cells using FACS were performed.
As a result, GFP-expressing cells were clearly increased as compared with Comparative Examples 1 and 2 (FIGS. 1E and F).

Example 1: Production of retinal tissue using human ES cells (Wnt signal pathway inhibitor and Matrigel, serum addition conditions)
RAX :: GFP knock-in human ES cells (derived from KhES-1) were cultured according to the methods described in “Ueno, M. et al. PNAS 2006” and “Watanabe, K. et al. Nat Biotech 2007” and were used for experiments. Using. 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. Suspension culture was performed in 100 μl of serum-free medium at 37 ° C. and 5% CO ₂ so that 9 × 10 3 cells were obtained per well. 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. Furthermore, 1/10 amount of fetal calf serum per volume was added on the 12th day from the start of suspension culture. Thereafter, suspension culture was continued, and on the 18th day from the start of suspension culture, observation of the fluorescence microscope and confirmation of the ratio of GFP-expressing cells using FACS were performed. At the same time, the experiment was performed under the conditions of Comparative Example 3 in which serum was not added.
While the ratio of GFP-expressing cells was 3.2% (FIGS. 2A, B, and 3A) under the conditions of Comparative Example 3, many GFP-expressing cells appeared when serum was added (FIGS. 2C and D). . From the analysis using FACS, the percentage of GFP positive cells was slightly over 30% (FIG. 3B).

Example 2: Production of retinal tissue using human ES cells (Wnt signal pathway inhibitor and Matrigel, serum and Shh signal agent addition conditions)
RAX :: GFP knock-in human ES cells (derived from KhES-1) were cultured according to the methods described in “Ueno, M. et al. PNAS 2006” and “Watanabe, K. et al. Nat Biotech 2007” and were used for experiments. Using. 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. Suspension culture was performed in 100 μl of serum-free medium at 37 ° C. and 5% CO ₂ so that 9 × 10 3 cells were obtained per well. 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, 1/10 amount of fetal calf serum and Shh signal pathway agonist (100 nM SAG) per volume were added and suspension culture was performed. On the 18th day from the start of suspension culture, the ratio of GFP-expressing cells was examined using FACS.
By adding an Shh signal pathway agonist at the same time as serum, a large number of GFP-expressing cells appeared (FIGS. 2E, F). From the analysis using FACS, the percentage of GFP-expressing cells reached 70% or more.
Compared to Comparative Example 3, the ratio of GFP-expressing cells is about 10 times higher under the conditions of Example 1 where serum is added, and the ratio of GFP-expressing cells is higher under the conditions of Example 2 where serum and Shh signal pathway agonists are added simultaneously. Was found to increase about 24 times (FIG. 3D).

Example 3: Confirmation of formation of retinal tissue (Method)
The aggregates having GFP-expressing cells produced in Examples 2 and 3 were confirmed for retinal tissue formation. From the 18th day of suspension culture, suspension culture was performed under 40% O ₂ using DMEM / F12 medium supplemented with N2 supplement, 10% (v / v) fetal bovine serum, and 0.5 μM retinoic acid. It was. Thereafter, the aggregate was fixed with a 4% paraformaldehyde solution, a frozen section was prepared, and the tissue structure was confirmed by immunostaining.
As a result, on the 60th day from the start of suspension culture, Brn3 and TuJ1 positive ganglion cells in the bottom layer, Crx and Recoverin positive photoreceptor precursor cells in the outermost layer and intermediate layer, Brn3 positive cells in the outermost layer and the bottom layer It was revealed that interneuron progenitor cells such as Chx10-positive bipolar cells were arranged in an orderly manner between Brn3-positive cells (FIGS. 4A, B, and C). Furthermore, if suspension culture is continued until day 126, Crx and Recoverin-positive photoreceptor precursor cells accumulate in the outermost layer, and are specifically expressed in Nrl-expressing cells and pyramidal cells that are specifically expressed in rod cells. Cells expressing Rxr-gamma were observed. In the intermediate layer, cells expressing Ptf1a, a progenitor cell marker for horizontal cells and amacrine cells, were observed (FIGS. 4D, E, F, G, H, and I). From this result, it was shown that retinal tissue can be produced with high efficiency from human ES cells.

Example 4: Transplantation of retinal tissue 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. A retinal tissue is transplanted into the formed space using a cell transplant needle or a cell sheet transplant device.

  According to the method of the present invention, it is possible to produce retinal tissue with high efficiency. The production method of the present invention is a method for efficiently producing cell groups (photocells, optic nerves, etc.) constituting a retinal tissue for the purpose of evaluating toxicity and drug efficacy of chemical substances, cell therapy, etc., and retinal tissue having a tissue structure. It is very useful from the viewpoint of efficiently producing retinal tissue, which is a "tissue material" for use in testing and treatment, for the purpose of evaluating toxicity and drug efficacy using glycine and applying it to transplantation materials for retinal tissue transplantation treatment. is there.

Claims (5)

The manufacturing method of the retinal tissue characterized by including the process of following (1)-(3).
(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. The aggregate formed is suspended in a serum-free medium containing a basement membrane preparation and a serum substitute . Second step (3) The aggregate cultured in the second step is suspended in a serum medium . Third step to induce retinal progenitor cells The method for producing retinal tissue according to claim 1, wherein the pluripotent stem cells are primate pluripotent stem cells. The method for producing retinal tissue according to claim 1 or 2, wherein the pluripotent stem cells are human pluripotent stem cells. The retinal tissue according to any one of claims 1 to 3, 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. Manufacturing method. The method for producing retinal tissue according to any one of claims 1 to 4, wherein the first to third steps are performed in the presence of a knockout serum substitute.

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