WO2017043605A1 - 網膜色素上皮細胞の製造方法 - Google Patents
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Definitions
- the present invention relates to a method for producing retinal pigment epithelium (RPE) cells and the like.
- RPE retinal pigment epithelium
- Retinal pigment epithelial cells are pigment epithelial cells present in the outermost layer of the retina, and play an important role in the maintenance of photoreceptor cells such as phagocytosis of photoreceptor outer segments and recycling of visual substances.
- Age-related macular degeneration caused by abnormalities of retinal pigment epithelial cells due to aging or the like is an eye disease that causes a decrease in central vision and blindness, and the development of an effective treatment method is desired.
- cell transplantation therapy that replenishes and replaces retinal pigment epithelial cells has attracted attention as a new treatment method for age-related macular degeneration, and retinal pigment epithelial cells are expected to be used as transplantation materials for cell therapy.
- Non-patent Documents 1, 2, Patent Documents 1, 2, 3, 4 There have been several reports (Non-patent Documents 1, 2, Patent Documents 1, 2, 3, 4) regarding methods for inducing differentiation of retinal pigment epithelial cells using human pluripotent stem cells. In the industry, a technique for producing high-quality retinal pigment epithelial cells stably and in large quantities is required, and a more efficient and simple production method has been desired.
- retinal pigment epithelial cells derived from pluripotent stem cells are used for regenerative medicine and the like, in order to stably produce the cells in large quantities, it is urgent to develop a more efficient production method.
- the present invention relates to the following.
- a method for producing retinal pigment epithelial cells comprising the following steps: (1) a first step of culturing pluripotent stem cells in a medium containing an FGF receptor inhibitor and / or a MEK inhibitor for a period not exceeding 30 days, and (2) the cells obtained in the first step A second step of culturing in the presence of a Nodal signaling pathway inhibitor and / or a Wnt signaling pathway inhibitor to form retinal pigment epithelial cells.
- the production method according to [1] wherein the first step is performed under serum-free conditions.
- the medium in the first step further contains a sonic hedgehog signal transduction pathway agent.
- the sonic hedgehog signal transduction pathway agent is SAG.
- the medium in the first step further contains a PKC inhibitor.
- the PKC inhibitor is Go6983.
- the culture period is a period that is sufficient to induce expression of at least one gene of an ocular transcription factor and does not exceed 30 days. The manufacturing method in any one.
- the culture period is a period sufficient to induce expression of at least one gene of PAX6, LHX2 and SIX3 and not exceeding 30 days
- the manufacturing method in any one of. [21] The production method according to any one of [1] to [20], [21 ′] and [22 ′] above, wherein the culture period is 2 days to 13 days in the first step. [22] The production method according to any one of [1] to [21], [21 ′] and [22 ′] above, wherein the culture period is 4 to 6 days in the first step.
- test substance comprising retinal pigment epithelial cells produced by the method according to any one of [1] to [22], [21 ′] and [22 ′] above reagent.
- a test substance is brought into contact with retinal pigment epithelial cells produced by the method according to any one of [1] to [22], [21 ′] and [22 ′], and the substance affects the cells.
- a method for evaluating the toxicity / drug efficacy of the substance, comprising testing the effect.
- a pharmaceutical composition comprising a retinal pigment epithelial cell produced by the method according to any one of [1] to [22], [21 ′] and [22 ′] as an active ingredient.
- the present invention has made it possible to provide a method for producing retinal pigment epithelial cells with higher efficiency than existing differentiation induction methods. Therefore, the method of the present invention is useful from the viewpoint of efficiently producing retinal pigment epithelial cells that are used as a cell therapy transplant material or a reagent / material used for toxicity / drug efficacy evaluation of chemical substances and the like.
- IPS cells (201B7 or 1231A3) -derived retinal pigment epithelium (RPE) cells manufactured using "StemFit (registered trademark)" AK03 medium or Essential 8 medium with a MEK inhibitor treatment step Of 6-well culture plates on days 38-42 of culture containing MEKi: MEK inhibitor (when using “StemFit (registered trademark)” AK03 medium: 1 ⁇ M, when using Essential-8 medium: 0.03 ⁇ M PD0325901).
- Photograph (a), low-magnification phase-contrast microscope image (b), high-magnification image of 55-day culture plate containing iPS cell (201B7) -derived RPE cells produced by a production method that includes a MEK inhibitor treatment process
- Bright field microscope image (c).
- MEKi MEK inhibitor (1 ⁇ M PD0325901).
- An iPS cell (201B7, or 201B7 manufactured using a StemFit (registered trademark) AK03 medium, an Essential 8 medium, or StemSure hPSC Medium ⁇ w / o bFGF in a production method including an FGF receptor inhibitor treatment step , 1231A3) Photo of a 6-well culture plate on day 38-42 of culture containing retinal pigment epithelium (RPE) cells.
- FGFRi FGF receptor inhibitor (100 nM PD173074).
- FGFRi FGF receptor inhibitor (100 nM PD173074).
- IPS cells (201B7 or 1231A3) produced by a production method comprising a combination treatment step of a MEK inhibitor and / or FGF receptor inhibitor and various inhibitors, signal transduction pathway inhibitors or signal transduction pathway agonists
- MEKi MEK inhibitor (1 ⁇ M PD0325901)
- FGFRi FGF receptor inhibitor (100 nM PD173074)
- BMPPRi BMP receptor inhibitor (100 nM LDN193189)
- Shh ag Shh signaling pathway agonist (30 nM SAG )
- PKCi PKC inhibitor (2 M Go6983).
- a photograph of a typical 6-well culture plate at each stage when divided into 6 stages from 0 to 5 depending on the proportion of RPE cells in the whole well (A), production method not including MEK inhibitor treatment process (No treatment), production method including MEK inhibitor treatment process (MEKi), and production method (MEKi + PKCi, MEKi +) including combination treatment step of MEK inhibitor and various inhibitors or signal transduction pathway inhibitors Summary of results (B) produced by PKCi + BMPRi, MEKi + FGFRi, MEKi + FGFRi + BMPRi, MEKi + FGFRi + PKCi, MEKi + FGFRi + PKCi + BMPRi), and does not include FGF receptor inhibitor treatment step Manufacturing method (no treatment), manufacturing method including FGF receptor inhibitor treatment step (FGFRi), and manufacturing method including combination treatment step of FGF receptor inhibitor and various inhibitors or signal transduction pathway inhibitors ( FGFRi + PKCi, FGFRi + PKCi + BMPRi, FGFRi +
- the vertical axis of the graph displays the ratio of RPE cells in the whole well in 6 levels. Values are mean ⁇ standard deviation, and n indicates the number of experiments.
- MEKi MEK inhibitor (1 M PD0325901)
- FGFRi FGF receptor inhibitor (100 nM PD173074)
- BMPPRi BMP receptor inhibitor (100 nM LDN193189)
- PKCi PKC inhibitor (2 ⁇ M Go6983).
- Retinal pigment epithelium derived from iPS cells (Ff-I01 or QHJI01) produced using a “StemFit (registered trademark)” AK03N medium in a production method including a MEK inhibitor or FGF receptor inhibitor treatment step )
- a photograph of a 6-well culture plate on day 43 of culture containing iPS cell-derived differentiated cells produced by a production method that does not include a MEK inhibitor and / or FGF receptor inhibitor treatment step is also shown (no treatment). .
- MEKi MEK inhibitor (1 ⁇ PD0325901)
- FGFRi FGF receptor inhibitor (100 nM PD173074).
- a culture method comprising a retinal pigment epithelium (RPE) cell derived from an iPS cell (QHJI01) produced using a “StemFit (registered trademark)” AK03N medium in a production method including a MEK inhibitor or FGF receptor inhibitor treatment step 48 Photograph of 6-well culture plate on day (MEKi (PD0325901) or FGFRi (PD173074)). The effects of inhibitor exposure days from 1 to 6 days were examined.
- MEKi MEK inhibitor (1 ⁇ PD0325901)
- FGFRi FGF receptor inhibitor (100 nM PD173074).
- IPS cells Ff-I01 or QHJI01 on the 43rd day of culture, produced using “StemFit (registered trademark)” AK03N medium, with MEK inhibitor or FGF receptor inhibitor treatment step
- Results of visual determination of the proportion of retinal pigment epithelium (RPE) cells in the whole well according to FIG. 8A, and the expression values (Signal) and flags (Detection) of PAX6, LHX2, and SIX3 at the end of the first step are shown ( MEKi or FGFRi).
- the visual determination result of the ratio of RPE cells in the whole well of iPS cell-derived differentiated cells produced by a production method that does not include a MEK inhibitor and / or FGF receptor inhibitor treatment step, and the end of the first step The expression values (Signal) and flags (Detection) of PAX6, LHX2, and SIX3 at the time corresponding to time are shown (no treatment).
- MEKi MEK inhibitor (1 ⁇ PD0325901)
- FGFRi FGF receptor inhibitor (100 nM PD173074).
- a culture method comprising a retinal pigment epithelium (RPE) cell derived from an iPS cell (QHJI01) produced using a “StemFit (registered trademark)” AK03N medium in a production method including a MEK inhibitor or FGF receptor inhibitor treatment step 36
- RPE retinal pigment epithelium
- MEKi MEK inhibitor (PD0325901)
- FGFRi FGF receptor inhibitor (PD173074).
- a method comprising a MEK inhibitor or FGF receptor inhibitor treatment step, and a culture containing iPS cells (QHJI01) -derived retinal pigment epithelium (RPE) cells produced using "StemFit (registered trademark)" AK03N medium 49 Photograph of 6-well culture plate on day (MEKi (PD0325901) or FGFRi (PD173074)).
- MEKi MEK inhibitor (1 ⁇ M PD0325901)
- FGFRi FGF receptor inhibitor (100 nM PD173074).
- iPS cell QHJI01 or 1231A3 -derived retinal pigment epithelium (RPE) cells produced using "StemFit (registered trademark)" AK03N medium in a production method that includes a MEK inhibitor treatment step , Photographs of 6-well culture plates on day 50 (MEKi (PD0325901), MEKi (PD184352), MEKi (U0126), MEKi (TAK-733), or MEKi (AZD-8330)). For comparison, a photograph of a 6-well culture plate on day 49 of culture containing iPS cell-derived differentiated cells produced by a production method that does not include a MEK inhibitor treatment step is also shown (no treatment).
- MEKi MEK inhibitor (1 ⁇ M259PD0325901, 1.5 ⁇ M, 3 ⁇ M, 6 ⁇ PD184352, 5 ⁇ M, 10 ⁇ U0126, 0.3 ⁇ M TAK-733, 0.3 ⁇ M AZD-8330).
- a culture method comprising a retinal pigment epithelium (RPE) cell derived from an iPS cell (QHJI01 or 1231A3) produced using a “StemFit (registered trademark)” AK03N medium in a production method including an FGF receptor inhibitor treatment step 49 Photograph of 6-well culture plate on day (FGFRi (PD173074) or FGFRi (SU5402)).
- RPE retinal pigment epithelium
- FGFRi An FGF receptor inhibitor (100 nM PD173074, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M SU5402).
- NODALi Nodal signaling pathway inhibitor (5 ⁇ M SB431542)
- WNTi Wnt signaling pathway inhibitor (3 ⁇ M CKI-7).
- RPE retinal pigment epithelium
- the visual determination result of the ratio of RPE cells in the whole well of iPS cell-derived differentiated cells on the 39th day of culture produced by a production method that does not include a MEK inhibitor and / or BMP receptor inhibitor treatment step (
- the expression levels of BEST1, MITF, and RAX are also shown (bottom in the figure, the ratio of RPE cells occupying the whole well (6-step display) “1”).
- the gene expression level of each sample was corrected with the expression level of GAPDH, and in the comparison of the expression levels of BEST1, MITF, and RAX, the expression of iPS cells that were cultured under undifferentiated maintenance culture conditions (lower in the figure, undifferentiated) The relative amount when the amount is 1 is shown.
- a culture method comprising a retinal pigment epithelium (RPE) cell derived from an iPS cell (1231A3) produced using a “StemFit (registered trademark)” AK03N medium in a production method comprising a MEK inhibitor or FGF receptor inhibitor treatment step 43 Photograph of 6-well culture plate on day (right side of the figure, MEKi (PD0325901), FGFRi (PD173074)) and retinal pigment epithelial markers RPE65, BEST1, CRALBP, endogenous control GAPDH on culture day 43 by RT-PCR method The results of confirming the expression (MEKi (PD0325901), FGFRi (PD173074) on the left side of the figure).
- RPE retinal pigment epithelium
- a photograph of a 6-well culture plate on day 43 of culture containing iPS cell-derived differentiated cells produced by a production method that does not include a MEK inhibitor and / or FGF receptor inhibitor treatment step (right side of the figure, no Treatment) and RT-PCR results of RPE65, BEST1, CRALBP, and GAPDH are also shown (left side in the figure, no treatment).
- RT-PCR primary human RPE (left side in the figure, hRPE) was used, and iPS cells cultured under undifferentiated maintenance culture conditions (left side in the figure, undifferentiated iPSC) were used as a negative control.
- a “pluripotent stem cell” is a cell having self-renewal ability and pluripotency, can be cultured in vitro, and has three germ layers (ectodermal, mesoderm, endoderm). A stem cell having the ability (pluripotency) to differentiate into all cell lineages to which it belongs.
- pluripotent stem cells examples include embryonic stem cells (ES cells: Embryonic stem cells) and induced pluripotent stem cells (iPS cells: induced pluripotent stem cells).
- the pluripotent stem cell used in the present invention is a mammalian pluripotent stem cell, preferably a rodent or primate pluripotent stem cell, and more preferably a human pluripotent stem cell.
- mammals include primates such as humans and monkeys, rodents such as mice, rats, hamsters, and guinea pigs, and other dogs, cats, pigs, cows, goats, horses, sheep, rabbits, and the like.
- Embryonic stem cells can be produced, for example, by culturing an inner cell mass present in a blastocyst stage embryo before implantation on a feeder cell or in a medium containing LIF. Specific methods for producing embryonic stem cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718 and the like. Embryonic stem cells can be obtained from a predetermined institution or a commercial product can 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, both mouse embryonic stem cells, are available from RIKEN, and the D3 strain is available from ATCC.
- ntES cells Nuclear transfer ES cells
- ntES cells which are one of ES cells, can be established from cloned embryos made by transplanting somatic cell nuclei into eggs from which the cell nuclei have been removed.
- “Artificial pluripotent stem cells” are cells in which pluripotency is induced by reprogramming somatic cells by a known method or the like. Specifically, for example, somatic cells such as fibroblasts, skin cells, peripheral blood mononuclear cells, Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Examples include cells that have been reprogrammed and induced pluripotency by introducing any of a plurality of recombination factor combinations selected from the gene group such as Sall4, lin28, and Esrrb.
- somatic cells such as fibroblasts, skin cells, peripheral blood mononuclear cells, Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog.
- somatic cells such as fibroblasts, skin cells, peripheral blood mononuclear cells, Oct3 / 4, Sox2, K
- Preferred combinations of reprogramming factors include (1) Oct3 / 4, Sox2, Klf4 and Myc (c-Myc or L-Myc), (2) Oct3 / 4, Sox2, Klf4, Lin28 and L-Myc (Stem Cells, 2013; 31: 458-466).
- Artificial pluripotent stem cells were first established in mouse cells in 2006 by Yamanaka et al. (Cell, 2006, 126 (4) pp. 663-676), and in 2007 were also established in human fibroblasts (Cell, 2007, 131 (5) pp.861-872; Science, 2007, 318 (5858) pp.1917-1920; Nat. Biotechnol., 2008, 26 (1) pp.101-106).
- induced pluripotent stem cells can also be induced from somatic cells by the addition of compounds (Science, 2013, 341 pp. 651-654 ).
- artificial pluripotent stem cells such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells established at Kyoto University
- human induced pluripotent cell lines such as 1231A3 cells are available from Kyoto University or iPS Academia Japan.
- established artificial pluripotent stem cells for example, Ff-I01 cells, Ff-I14 cells and QHJI01 cells established at Kyoto University are available from Kyoto University.
- Fibroblasts include those derived from the dermis.
- the means for gene expression is not particularly limited, and gene transfer methods well known to those skilled in the art or direct protein An injection method can be used.
- the gene introduction method include infection methods using viral vectors (eg, retrovirus vectors, lentivirus vectors, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors), plasmid vectors (eg, plasmid vectors, Episomal vector), or the calcium phosphate method, lipofection method, retronectin method, electroporation method and the like using RNA vectors.
- induced pluripotent stem cells When producing induced pluripotent stem cells, they can be produced in the presence of feeder cells or in the absence of feeder cells (feeder-free). When producing an induced pluripotent stem cell in the presence of a feeder cell, the induced pluripotent stem cell can be produced in the presence of an undifferentiated maintenance factor by a known method.
- the medium used for producing the induced pluripotent stem cells in the absence of feeder cells is not particularly limited. However, a known embryonic stem cell and / or artificial pluripotent stem cell maintenance medium or feeder-free artificial medium can be used. A medium for establishing pluripotent stem cells can be used.
- a medium for establishing feeder-free artificial pluripotent stem cells for example, Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, StabilizedsEssential 8 medium, StemFit (trademark) And the like.
- E8 medium Essential 8 medium
- TeSR medium TeSR medium
- mTeSR medium mTeSR-E8 medium
- StabilizedsEssential 8 medium StemFit (trademark) And the like.
- StemFit trademark
- Pluripotent stem cells can be generated.
- the pluripotent stem cells used in the present invention are preferably rodent or primate induced pluripotent stem cells, and more preferably human induced pluripotent stem cells.
- Pluripotent stem cells can be maintained and expanded by methods known to those skilled in the art. However, from the viewpoint of the safety of transplanted cell production, pluripotent stem cells are used under serum-free conditions and feeder cells. It is preferable to perform maintenance culture and expansion culture in the absence.
- a genetically modified pluripotent stem cell can be prepared, for example, by using a homologous recombination technique.
- genes on the chromosome to be modified include cell marker genes, histocompatibility antigen genes, and disease-related genes based on retinal pigment epithelial cell disorders. Modification of target genes on chromosomes can be found in Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Gene Targeting, A Practical Approach, roIRL Press at Oxford University Press (1993); 8, gene targeting, production of mutant mice using ES cells, Yodosha (1995); and the like.
- genomic DNA containing a target gene to be modified (for example, cell marker gene, histocompatibility antigen gene, disease-related gene, etc.) is isolated, and the target gene is identified using the isolated genomic DNA.
- a target vector for homologous recombination is prepared. 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 in which the gene on the chromosome is modified can be prepared.
- Genomic DNA containing the target gene can also be isolated by using a genomic DNA library screening system (Genome System) or Universal Genome Walker Kits (CLONTECH). Instead of genomic DNA, a polynucleotide encoding the target protein can also be used. The polynucleotide can be obtained by amplifying the corresponding polynucleotide by PCR.
- the target vector can be either a replacement type or an insertion type.
- methods such as positive selection, promoter selection, negative selection, or poly A selection can be used. Examples of a method for selecting a target homologous recombinant from the selected cell line include Southern hybridization method and PCR method for genomic DNA.
- Eye-forming transcription factor (Eye ⁇ ⁇ Field Transcription ⁇ Factors)” in the present invention is a gene expressed in the eye formation region in the early stage of development, and is ET (Tbx3), Rx1 (Rax), Pax6, Six3, Lhx2, Tlx (Nr2e1 ), Optx2 (Six6) and the like have been identified. These eye formation transcription factors can be used as a marker at the early stage of eye formation.
- the “retinal pigment epithelial cell” in the present invention means an epithelial cell existing outside the neural retinal tissue in the living retina. Whether or not the cells are retinal pigment epithelial cells can be determined by those skilled in the art, for example, expression of cell markers (RPE65, Mitf, CRALBP, MERTK, BEST1, etc.), presence of melanin granules (dark brown), tightness between cells It can be easily confirmed by junctions, polygonal and paving stone characteristic cell morphology. Whether or not the cells have the function of retinal pigment epithelial cells can be easily confirmed by the secretion ability of cytokines such as VEGF and PEDF.
- cytokines such as VEGF and PEDF.
- the “floating culture” in the present invention is a culture carried out under conditions that maintain a state in which cells or cell aggregates are suspended in a culture solution, that is, between cells or cell aggregates and a culture device or the like. Cultivation under conditions that do not allow the formation of strong cell-substratum junctions.
- “Adhesion culture” refers to a culture performed under conditions in which cells or cell aggregates adhere to a culture apparatus or the like. In this case, the term “cell adheres” means that a strong cell-substrate bond can be formed between the cell or the cell aggregate and the culture equipment. That is, the adhesion culture refers to culture under conditions that cause a strong cell-substrate bond between cells or cell aggregates and culture equipment.
- the cells are plane-attached.
- cell aggregates in suspension culture cell-matrix bonds are hardly formed between the culture equipment and the like, or even if formed, the contribution is small.
- the intrinsic cell-substrate bond is present inside the aggregate, but the cell-substrate bond is hardly formed between the culture apparatus and the like. Even if it is formed, its contribution is small.
- Cell-to-cell adhesion means that cells adhere to each other on the surface. More specifically, the term “cell-cell adhesion” means that the proportion of the surface area of one cell that adheres to the surface of another cell is, for example, 1% or more, preferably 3% or more, more preferably 5%.
- the surface of the cell can be observed by staining with a reagent that stains the membrane (eg, DiI) or immunostaining of a cell adhesion factor (eg, E-cadherin or N-cadherin).
- a reagent that stains the membrane eg, DiI
- a cell adhesion factor eg, E-cadherin or N-cadherin
- the incubator used for the adhesion culture is not particularly limited as long as it can “adherently culture”, and a person skilled in the art can appropriately incubate according to the culture scale, culture conditions, and culture period. Can be selected.
- Examples of such an incubator include a tissue culture flask, a culture dish (dish), a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multiplate, a multiwell plate, a chamber slide, a petri dish, a tube, Examples include trays, culture bags, microcarriers, beads, spinner flasks or roller bottles.
- These incubators are preferably cell adhesive in order to enable adhesion culture.
- Examples of the cell adhesive incubator include an incubator in which the surface of the incubator is artificially treated for the purpose of improving the adhesion with cells, and specifically, a culture in which the inside is coated with a coating agent. Vessel.
- Examples of the coating agent include laminin [including laminin ⁇ 5 ⁇ 1 ⁇ 1 (hereinafter referred to as laminin 511), laminin ⁇ 1 ⁇ 1 ⁇ 1 (hereinafter referred to as laminin 111), and laminin fragments (such as laminin 511E8), entactin, collagen, gelatin, vitronectin (Vitronectin), Examples include an extracellular matrix such as Synthemax (Corning) and Matrigel, or a polymer such as polylysine and polyornithine.
- a culture vessel having a surface processed such as a positive charge treatment can also be used. Since stable and efficient induction of retinal pigment epithelial cells is possible, an incubator coated with laminin 511E8 is more preferably used (WO2015 / 053375).
- Laminin 511E8 can be purchased commercially (eg, iMatrix-511, Nippi).
- 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, A petri dish, a tube, a tray, a culture bag, a spinner flask, or a roller bottle is mentioned.
- These incubators are preferably non-cell-adhesive in order to enable suspension culture.
- the surface of the incubator is artificially treated for the purpose of improving adhesion to cells (for example, an extracellular matrix such as laminin, entactin, collagen, gelatin, or polylysine)
- a coating treatment with a polymer such as polyornithine or a surface treatment such as a positive charge treatment can be used.
- the surface of the incubator was artificially treated (for example, super hydrophilic treatment such as MPC polymer, low protein adsorption treatment, etc.) for the purpose of reducing the adhesion to cells. Things can be used.
- the medium used for cell culture can be prepared using a medium usually used for animal cell culture as a basal medium.
- basal media include, for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium , F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, and the like. These can be used alone or in combination of two or more, but are not limited thereto.
- the “serum medium” in the present invention means a medium containing unadjusted or unpurified serum.
- serum derived from any animal can be used, serum derived from mammals such as cows and humans can be preferably used.
- the patient's own serum can also be used.
- the serum concentration is not particularly limited as long as it can induce differentiation of retinal pigment epithelial cells efficiently, but can be appropriately set within a range of, for example, about 0.5% to 30% (v / v).
- concentration may be fixed and may be changed in steps.
- “Serum-free medium” means a medium that does not contain unconditioned or unpurified serum.
- a medium containing purified blood-derived components or animal tissue-derived components (for example, growth factors) is also included in the serum-free medium unless it contains unconditioned or unpurified serum.
- “Serum-free condition” means a condition that does not contain unadjusted or unpurified serum, specifically, a condition using a serum-free medium.
- the serum-free medium may contain a serum replacement.
- serum substitutes include those containing albumin, transferrin, fatty acid, collagen precursor, trace elements, 2-mercaptoethanol or 3 ′ thiolglycerol, or their equivalents as appropriate.
- serum replacement can be prepared, for example, by the method described in WO98 / 30679.
- a commercially available product may be used as a serum substitute.
- examples of such commercially available serum substitutes include Knockout TM Serum Replacement (manufactured by Life Technologies: hereinafter referred to as KSR), Chemically-defined Lipid concentrate (manufactured by Life Technologies), Glutamax TM (manufactured by Life Technologies). Manufactured), B27 (manufactured by Life Technologies), and N2 (manufactured by Life Technologies).
- an appropriate amount for example, about 0.5% to about 30%, preferably about 5% to about 20%
- a commercially available KSR manufactured by Life Technologies
- Added serum-free medium for example, a medium obtained by adding KSR in the above concentration range to GlasgowgoMEM medium
- the medium used in the present invention appropriately contains fatty acids or lipids, amino acids (eg, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like. May be.
- the medium used in the present invention contains a ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor for the purpose of suppressing cell death (apoptosis) of pluripotent stem cells.
- ROCK inhibitors include Y-27632, Fasudil, or H-1152.
- concentration of the ROCK inhibitor can be appropriately set by those skilled in the art, and can be set, for example, from a range of concentrations exhibiting ROCK inhibitory activity corresponding to about 50 nM to 200 ⁇ M of Y-27632.
- the medium used in the present invention is preferably a medium (Chemically defined medium; medium CDM) in which the components are chemically determined from the viewpoint of avoiding mixing of chemically undecided components.
- medium CDM Chemical defined medium
- the culture in the present invention is preferably performed under xenofree conditions.
- “Zeno-free” means a condition in which a component (protein or the like) derived from a biological species different from the biological species of the cell to be cultured is excluded.
- a medium containing substance X and “in the presence of substance X” means a medium to which exogenous substance X is added, a medium containing exogenous substance X, or exogenous substance X. Means in the presence of That is, when cells existing in the medium express, secrete or produce the substance X endogenously, the endogenous substance X is distinguished from the foreign substance X. It is understood that the medium that does not contain the substance X does not fall under the category of “medium containing substance X” even if it contains the endogenous substance X.
- the “medium containing an FGF signal transduction pathway agonist” is a medium to which an exogenous FGF signal transduction pathway agonist is added or a medium containing an exogenous FGF signal transduction pathway agonist.
- feeder cells are cells other than the stem cells that coexist when pluripotent stem cells are cultured.
- the feeder cells used for the undifferentiated maintenance culture of pluripotent stem cells include mouse fibroblasts (MEF), human fibroblasts, SNL cells, and the like.
- a feeder cell subjected to growth inhibition treatment is preferable.
- the growth inhibitory treatment include treatment of a growth inhibitor (for example, mitomycin C) or gamma irradiation.
- Feeder cells used for undifferentiated maintenance culture of pluripotent stem cells are produced by secreting humoral factors (preferably undifferentiated maintenance factors) and creating scaffolds for cell adhesion (extracellular matrix). Contributes to maintaining undifferentiation.
- the absence of feeder cells means culturing in the absence of feeder cells.
- the absence of feeder cells includes, for example, conditions in which no feeder cells are added, or conditions in which feeder cells are substantially not included (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less). It is done.
- the production method of the present invention is a method for producing retinal pigment epithelial cells, comprising the following steps (1) to (2): (1) a first step of culturing pluripotent stem cells in a medium containing an FGF receptor inhibitor and / or a MEK inhibitor for a period not exceeding 30 days, and (2) the cells obtained in the first step A second step of culturing in the presence of a Nodal signaling pathway inhibitor and / or a Wnt signaling pathway inhibitor to form retinal pigment epithelial cells.
- Preferred pluripotent stem cells in the first step include induced pluripotent stem cells, more preferably human induced pluripotent stem cells.
- the method for producing induced pluripotent stem cells is not particularly limited, and can be produced by a method well known to those skilled in the art as described above. However, the method for producing induced pluripotent stem cells (that is, initializing somatic cells) It is desirable that the step of establishing pluripotent stem cells is also performed feeder-free.
- Pluripotent stem cells are usually subjected to the first step after maintenance culture and expansion culture.
- Maintenance / expansion culture of pluripotent stem cells can be performed by methods well known to those skilled in the art, and is preferably performed in the absence of feeder cells. Further, the maintenance / expansion culture of pluripotent stem cells can be carried out by adhesion culture or suspension culture, but is preferably carried out by adhesion culture.
- the undifferentiation maintenance factor is not particularly limited as long as it is a substance having an action of suppressing differentiation of pluripotent stem cells, but is usually an undifferentiation maintenance factor derived from a mammal. Since an undifferentiated maintenance factor can have cross-reactivity between mammalian species, any mammalian undifferentiated maintenance factor can be used as long as the undifferentiated state of the pluripotent stem cells to be cultured can be maintained. However, preferably, an undifferentiated maintenance factor of a mammal of the same species as the cell to be cultured is used.
- FGF signal transduction pathway agents In the case of primed pluripotent stem cells (for example, human ES cells and human iPS cells), FGF signal transduction pathway agents, TGF ⁇ family signaling Examples include pathway agents. Specific examples of the FGF signal transduction pathway agent include fibroblast growth factors (for example, bFGF, FGF4, and FGF8). Examples of TGF ⁇ family signal transduction pathway agents include TGF ⁇ signal transduction pathway agents (eg, TGF ⁇ 1, TGF ⁇ 2) and Nodal / Activin signal transduction pathway agents (eg, Nodal, Activin A, Activin B). When culturing human pluripotent stem cells (human ES cells, human iPS cells), the undifferentiation maintenance factor is preferably bFGF.
- the concentration of undifferentiated maintenance factor in the medium used for maintenance / expansion culture of pluripotent stem cells is a concentration that can maintain the undifferentiated state of pluripotent stem cells, and can be appropriately set by those skilled in the art. it can.
- the concentration is usually about 4 to 500 ng / mL, preferably about 10 to 200 ng / mL, more preferably about 30 to 150 ng / mL.
- a medium containing an undifferentiated maintenance factor (hereinafter sometimes referred to as feeder-free medium)
- a medium commercially available as a medium for stem cells can be used as appropriate, for example, Essential 8 (manufactured by Life Technologies), hESF9 (Proc Natl Acad Sci U S A. 2008 Sep 9; 105 (36): 13409-14), S-medium (DS Pharma Biomedical), StemPro (Life Technologies), mTeSR1 (STEMCELL Technologies) ), MTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), and the like are commercially available.
- Essential 8 manufactured by Life Technologies
- hESF9 Proc Natl Acad Sci U S A. 2008 Sep 9; 105 (36): 13409-14
- S-medium DS Pharma Biomedical
- StemPro Life Technologies
- mTeSR1 StemTeSR1 (STEMCELL Technologies)
- MTeSR2
- StemFit registered trademark (manufactured by Ajinomoto Co., Inc.) can be mentioned as a feeder-free medium under development. By using these media, maintenance culture and expansion culture of pluripotent stem cells can be performed.
- the dispersed pluripotent stem cells are prepared by a dispersion operation.
- the dispersion operation of pluripotent stem cells may include mechanical dispersion treatment, cell dispersion treatment, and cytoprotective agent addition treatment. You may perform combining these processes.
- the cell dispersion treatment is performed simultaneously with the cell protective agent addition treatment, and then the mechanical dispersion treatment is performed.
- the cytoprotective agent used in the cytoprotective agent addition treatment include heparin, serum, or serum substitute.
- a ROCK inhibitor may be added during dispersion.
- ROCK inhibitors examples include Y-27632, Fasudil (HA1077), H-1152, and the like.
- Examples of the cell dispersion used for the cell dispersion treatment include solutions containing any of enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase or papain, and a chelating agent such as ethylenediaminetetraacetic acid. .
- Commercially available cell dispersions such as TrypLE Select (Life Technologies) or TrypLE Express (Life Technologies) can also be used.
- Examples of the mechanical dispersion treatment include pipetting or scraping with a scraper.
- the dispersed pluripotent stem cells can be seeded in a new culture container and used for the first step.
- pluripotent stem cells are seeded in a new culture vessel and then maintained in the presence of a ROCK inhibitor Then, the first step may be started thereafter.
- the period of the ROCK inhibitor treatment is not particularly limited as long as cell death induced by dispersion can be suppressed, but is usually about 12 to 24 hours.
- the concentration of pluripotent stem cells at the start of the first step can be appropriately set by those skilled in the art, but in the case of adhesion culture, for example, 1.0 ⁇ 10 2 cells / cm 2 to 1 ⁇ 10 6 cells / cm 2 , preferably 2.0 ⁇ 10 2 cells / cm 2 to 2 ⁇ 10 5 cells / cm 2 , more preferably 5 ⁇ 10 2 cells / cm 2 to 1 ⁇ 10 5 cells / cm 2 or 1 ⁇ 10 3 cells / cm 2 to 1 ⁇ 10 4 cells / cm 2 .
- the medium used in the first step is preferably a medium in which the components are chemically determined from the viewpoint of avoiding contamination with chemically undecided components.
- the medium used in the first step may be a serum medium or a serum-free medium, but is preferably a serum-free medium from the viewpoint of avoiding contamination with chemically undecided components.
- the medium used in the first step may contain a ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor for the purpose of suppressing cell death (apoptosis).
- ROCK inhibitors include Y-27632, Fasudil, or H-1152. Only one ROCK inhibitor may be used, or two or more ROCK inhibitors may be used in combination.
- concentration of the ROCK inhibitor can be appropriately set by those skilled in the art, and can be set, for example, from a range of concentrations exhibiting ROCK inhibitory activity corresponding to about 50 nM to 200 ⁇ M of Y-27632.
- the culture in the first step may be performed in the presence of feeder cells, but is preferably performed under conditions that do not include feeder cells from the viewpoint of avoiding contamination with chemically undecided components.
- the medium used in the first step may or may not contain an exogenous undifferentiation maintenance factor regardless of the culture in the absence of feeder cells or the culture in the presence of feeder cells.
- the first step of the present invention is more preferably performed in a medium containing an undifferentiated maintenance factor in the absence of feeder cells.
- the undifferentiation maintenance factor is not particularly limited as long as it is a substance having an action of suppressing differentiation of pluripotent stem cells, but preferably contains an FGF signal transduction pathway acting substance, more preferably bFGF.
- the undifferentiated maintenance factor concentration in the medium used in the first step may be within the concentration range of the undifferentiated maintenance factor when used in the maintenance culture / expansion culture of pluripotent stem cells.
- concentration is usually about 4 to 500 ng / mL, preferably 10 to 200 ng / mL, more preferably about 30 to 150 ng / mL.
- the medium used in the first step contains an FGF receptor inhibitor and / or MEK inhibitor. As long as the medium does not significantly reduce the production efficiency of retinal pigment epithelial cells by the production method of the present invention (for example, to the same level or lower than the efficiency when the first step is not performed), additional components (for example, any An action or inhibitor of a signal transduction pathway).
- the medium may further contain a BMP receptor inhibitor, a sonic hedgehog signaling pathway agonist, or a PKC inhibitor alone or in any combination of the following (1) to (4): Good: (1) BMP receptor inhibitor and sonic hedgehog signaling pathway agonist, (2) BMP receptor inhibitor and PKC inhibitor, (3) Sonic hedgehog signaling pathway agonist and PKC inhibitor (4) BMP receptor inhibitor, sonic hedgehog signaling pathway agonist, and PKC inhibitor.
- the FGF receptor inhibitor is not particularly limited as long as it can suppress signal transduction mediated by FGF, and may be any of protein, nucleic acid, and low molecular weight compound.
- FGF forms a family consisting of at least 22 types.
- Representative FGF receptors include FGFR1, FGFR2, FGFR3, and FGFR4.
- An FGF receptor inhibitor is a substance that inhibits one, a plurality, or all of them.
- Such substances include substances that directly act on FGF or FGF receptors (eg, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding FGF or FGF receptors (eg, antisense oligonucleotides, siRNA, etc.), Substances that inhibit the binding of FGF receptor to FGF (for example, soluble FGF receptor, FGF antagonist, etc.), substances that inhibit physiological activity resulting from signal transduction by FGF receptor [for example, FGF receptor by ATP competition Inhibits tyrosine kinase activity of PD173074 (N- [2-[[4- (Diethylamino) butyl] amino] -6- (3,5-dimethoxyphenyl) pyrido [2,3-d] pyrimidin-7-yl]- N '-(1,1-dimethylethyl) urea) or SU5402 (2-[(1,2-Dihydro-2-oxo-3H-indol-3
- PD173074 and SU5402 are known FGF receptor inhibitors, and commercially available products can be obtained as appropriate.
- Preferred examples of the FGF receptor inhibitor include PD173074 and SU5402.
- the concentration of the FGF receptor inhibitor contained in the culture medium is not particularly limited as long as retinal pigment epithelial cells can be produced by the method of the present invention, and those skilled in the art will depend on the type of FGF receptor inhibitor. It can be set appropriately.
- the concentration of the FGF receptor inhibitor is a concentration corresponding to the FGF receptor inhibitory activity of PD173074 1 to 1000 nM, preferably 10 to 500 nM, more preferably 25nM to 400nM, particularly preferably 30 to 300 nM.
- a range is mentioned.
- the concentration range is 0.1 to 500 ⁇ M, preferably 1 to 100 ⁇ M, more preferably 5 to 20 ⁇ M.
- the concentration may be constant throughout the first step or may be changed stepwise.
- the MEK inhibitor is not particularly limited as long as it is a substance that inhibits the expression or activity of the MEK family, and may be any of protein, nucleic acid, and low molecular weight compound.
- MEK1, MEK2, MEK3, etc. are mentioned as typical MEK families, and MEK inhibitors are substances that inhibit the expression or activity of one, plural or all of these MEK families.
- Such substances include substances that suppress the expression of genes encoding various MEKs (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the enzymatic activity of various MEKs [PD0325901 (N-[(2R) -2, 3-Dihydroxypropoxy] -3,4-difluoro-2-[(2-fluoro-4-iodophenyl) amino] -benzamide), PD184352 ((2-[(2-Chloro-4-iodophenyl) amino] -N-cyclopropylmethoxy ) -3,4-difluorobenzamide), PD98059 (2'-Amino-3'-Methoxyflavone), U0126 (1,4-Diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] butadiene), MEK162 (5-[(4-Bromo-2-fluorophenyl) amino]
- PD0325901, PD184352, PD98059, U0126, MEK162, SL327, TAK-733, and AZD-8330 are known MEK inhibitors, and commercially available products can be appropriately obtained.
- Preferred examples of the MEK inhibitor include PD0325901, PD184352, U0126, TAK-733 and AZD-8330.
- the concentration of the MEK inhibitor contained in the medium is not particularly limited as long as mammalian retinal pigment epithelial cells can be produced by the method of the present invention, and those skilled in the art can appropriately determine the concentration of MEK inhibitor depending on the type of MEK inhibitor. Can be set.
- the concentration of the MEK inhibitor is PD0325901 0.001 to 10 ⁇ M, preferably 0.005 to 5 ⁇ M, more preferably 0.1 to 4 ⁇ M, more preferably 0.25 to 4 ⁇ M, and particularly preferably 0.25 to 2 ⁇ M. Is mentioned.
- PD184352 includes, for example, a concentration range of 0.01 to 20 ⁇ M, preferably 0.1 to 10 ⁇ M, and more preferably 1.5 to 6 ⁇ M.
- the concentration may be constant throughout the first step or may be changed stepwise.
- the BMP receptor inhibitor is not particularly limited as long as it can suppress signal transduction mediated by BMP, and may be any of protein, nucleic acid, and low molecular weight compound.
- typical BMPs include BMP2, BMP4, BMP7, GDF7, and the like.
- BMP receptor (BMPR) is type I receptor (ALK (activin-receptor-like-kinase) -1, ALK-2, ALK-3, ALK-6) and type II receptor (ActRII, BMPRII) It exists as a heterodimer.
- BMP receptor inhibitors include BMP or substances that directly act on BMP receptors (eg, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP or BMP receptors (eg, antisense oligonucleotides, siRNA, etc.) ), Substances that inhibit the binding of BMP receptor to BMP (eg, soluble BMP receptor, BMP antagonist, etc.), substances that inhibit physiological activity resulting from signal transduction by BMP receptor [LDN193189 (4- [6- (4-Piperazin-1-ylphenyl) pyrazolo [1,5-a] pyrimidin-3-yl] quinoline) or Dorsomorphin (6- [4- [2- (1-Piperidinyl) ethoxy] phenyl] -3- (4 -pyridinyl) -pyrazolo [1,5-a] pyrimidine) and the like], but is not limited thereto.
- BMP receptor inhibitors include BMP or substances that
- LDN193189 and Dorsomorphin are known inhibitors of BMPI-type receptors, and commercially available products can be appropriately obtained.
- Preferred examples of the BMP receptor inhibitor include LDN193189.
- a sonic hedgehog (hereinafter, sometimes referred to as Shh) signal transduction pathway agent is not particularly limited as long as it is a substance capable of enhancing signal transduction mediated by Shh. Any of low molecular weight compounds may be used.
- Shh signal transduction pathway agent is preferably SAG.
- the PKC inhibitor is not particularly limited as long as it is a substance that can inhibit the expression or activity of protein kinase C (PKC), and may be any of protein, nucleic acid, and low molecular weight compound.
- PKC protein kinase C
- PKC is a protein family composed of at least 10 kinds of isozymes
- a PKC inhibitor is a substance that inhibits the expression or activity of one, plural or all of them.
- Such substances include substances that suppress the expression of genes encoding PKC (eg, antisense oligonucleotides, siRNA, etc.), substances that inhibit the enzyme activity of PKC [eg, Go6983 (3- [1- [3- (Dimethylamino) propyl] -5-methoxy-1H-indol-3-yl] -4- (1H-indol-3-yl) -1H-pyrrole-2,5-dione), etc.]
- Go6983 is a known PKC inhibitor, and a commercially available product can be appropriately obtained.
- a preferred PKC inhibitor is Go6983.
- the concentration of the BMP receptor inhibitory substance includes a range of concentrations corresponding to BDN receptor inhibitory activity of LDN193189 1 to 1000 nM, preferably 10 to 500 nM, more preferably 30 to 300 nM.
- the concentration of the substance acting on the Shh signal transduction pathway includes a concentration range corresponding to the Shh signal transduction action of SAG 1 to 2000 nM, preferably 3 to 1000 nM, more preferably 10 to 500 nM.
- concentration of the PKC inhibitor examples include a range of concentrations corresponding to PKC inhibitory activity of Go6983 0.05 to 20 ⁇ M, preferably 0.2 to 10 ⁇ M, more preferably 0.5 to 5 ⁇ M.
- concentration may be constant throughout the first step or may be changed stepwise.
- the culture of pluripotent stem cells in the first step may be performed under any conditions of suspension culture or adhesion culture, but is preferably performed by adhesion culture.
- the incubator used for adhesion culture of pluripotent stem cells is not particularly limited as long as cell adhesion culture is possible, but a cell adhesion incubator is preferable.
- a cell adhesion incubator an incubator that has been artificially treated for the purpose of improving the adhesion with cells can be used.
- the incubator in which the above-mentioned interior is coated with a coating agent is mentioned.
- laminin As a coating agent, laminin [including laminin ⁇ 5 ⁇ 1 ⁇ 1 (laminin 511), laminin ⁇ 1 ⁇ 1 ⁇ 1 (laminin 111) and the like and laminin fragment (laminin 511E8 etc.)], enteractin, collagen, gelatin, vitronectin (Vitronectin), synthemax (Corning) ), An extracellular matrix such as Matrigel, or a polymer such as polylysine or polyornithine, and more preferably laminin 511E8 (WO2015 / 053375).
- Laminin 511E8 can be purchased commercially (eg, iMatrix-511, Nippi).
- the incubator used for suspension culture of pluripotent stem cells is not particularly limited as long as suspension culture of cells is possible, but is preferably non-cell-adhesive.
- the culture conditions such as the culture temperature and CO 2 concentration in the first step can be appropriately set.
- 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%.
- Medium exchange can be performed during the first step.
- the method of exchanging the medium is not particularly limited, and the whole amount of the original medium may be replaced with a new medium, or only a part of the original medium may be replaced with a new medium.
- calculate the final concentration of substances (MEK inhibitor, FGF receptor inhibitor, etc.) included in the first step and then change according to the ratio of the medium to be replaced.
- a new medium containing the substance in concentration can be prepared and replaced with the original medium.
- the final concentration of the substance included in the first step may be changed during the culture.
- the tool used for culture medium exchange operation is not specifically limited, For example, a pipetter, a micropipette, a multichannel micropipette, a continuous dispenser, etc. are mentioned.
- a pipetter a pipetter, a micropipette, a multichannel micropipette, a continuous dispenser, etc. are mentioned.
- a multichannel pipetman may be used.
- the number of days in the first step should be within a period in which the cells generated by exposing the FGF receptor inhibitor and / or MEK inhibitor to the pluripotent stem cells have the ability to differentiate into retinal pigment epithelial cells.
- the pluripotent stem cells in the first step are cultured for a period not exceeding 30 days. The period may vary depending on the pluripotent stem cell line used and the like, but is usually 2 days or more, preferably 3 days or more, more preferably 4 days or more.
- the pluripotent stem cells in the first step are preferably cultured for 2 days to 13 days or 2 days to 6 days, and more preferably 4 days to 6 days.
- the number of days in the first step may be determined using as a marker a specific marker that is expressed in a cell obtained by treating pluripotent stem cells with the FGF receptor inhibitor and / or MEK inhibitor.
- a marker a specific marker that is expressed in a cell obtained by treating pluripotent stem cells with the FGF receptor inhibitor and / or MEK inhibitor.
- PAX6 Paned box protein 6
- LHX2 LIM homeobox 2
- SIX3 SIX homeobox 3
- other early markers specifically, at least one gene expression of ocular transcription factor It is possible to shift to the second step after culturing the first step for a sufficient period of time to induce.
- the “period not exceeding 30 days” in the first step for example, “at least one marker for early eye formation, specifically, sufficient for inducing the expression of an ocular transcription factor and 30 days” And “a period sufficient to induce the expression of at least one gene of PAX6, LHX2 and SIX3 and not exceeding 30 days”.
- the upper limit of the culture period may be, for example, a period not exceeding 30 days, a period not exceeding 13 days, or a period not exceeding 6 days.
- a given culture period in a given culture condition is “a period sufficient to induce expression of at least one early eye marker, specifically an ocular transcription factor” or “at least one of PAX6, LHX2 and SIX3” Is sufficient period to induce one gene expression ”means that the expression of at least one of these genes is significantly detected in the cell population after culturing in that period in that condition compared to the untreated control. It can be determined by checking whether or not. Those skilled in the art can detect the expression of these genes by, for example, techniques such as Northern blot, RT-PCR, and microarray.
- PAX6, LHX2 and SIX3 are genes encoding an ophthalmic transcription factor that is expressed in the eye formation region in the early stage of development, and human genes include Genbank Accession No .: NM_001127612, Genbank Accession No. : NM_004789, Genbank Accession No .: NM_005413.
- Those genes in other animal species such as mice can be easily identified by those skilled in the art. For example, the genes are identified from the nucleotide sequences of the genes listed at http://www.ncbi.nlm.nih.gov. I can do things.
- a method for producing retinal pigment epithelial cells including the following steps. (1) a first step of culturing pluripotent stem cells in a medium containing an FGF receptor inhibitor and / or a MEK inhibitor for a period sufficient to induce expression of at least one gene of an ocular transcription factor; and (2) A second step of culturing the cells obtained in the first step in the presence of a Nodal signaling pathway inhibitor and / or a Wnt signaling pathway inhibitor to form retinal pigment epithelial cells.
- a method for producing retinal pigment epithelial cells including the following steps is provided. (1) a first step of culturing pluripotent stem cells in a medium containing an FGF receptor inhibitor and / or MEK inhibitor for a period sufficient to induce expression of at least one gene of PAX6, LHX2 and SIX3; And (2) a second step of culturing the cells obtained in the first step in the presence of a Nodal signaling pathway inhibitor and / or a Wnt signaling pathway inhibitor to form retinal pigment epithelial cells.
- the cells can be detached from the incubator and recovered.
- the method for detaching cells from the incubator is not particularly limited as long as it is a generally known method for detaching cells, but a cell detachment solution containing an enzyme such as trypsin can be used.
- a commercially available cell detachment solution [TrypLE®select (Life Technologies) etc.] can also be used.
- the detached and recovered cells are usually washed with PBS (Phosphate Buffered Saline) and / or the medium used in the second step and then used in the second step.
- the cells obtained in the first step can be passaged (maintenance culture), stored as a production intermediate of retinal pigment epithelial cells, or subjected to other treatments as long as their differentiation state and survival state are maintained.
- the method for cryopreserving the cells obtained in the first step is not particularly limited as long as the method is generally known as a method for cryopreserving cells.
- the cells obtained in the first step may be DMSO, glycerin or the like. It can be suspended and stored frozen in a medium containing a frost protection agent.
- a commercially available cell cryopreservation solution [StemCellBanker (Zenoaq)] can also be used.
- Second step The second step of culturing the cells obtained in the first step in the presence of a Nodal signaling pathway inhibitor and / or a Wnt signaling pathway inhibitor to form retinal pigment epithelial cells is described. To do.
- the cell concentration at the start of the second step can be appropriately set by those skilled in the art, but in the case of adhesion culture, for example, 1.0 ⁇ 10 2 cells / cm 2 to 2 ⁇ 10 6 cells / cm 2 , preferably 3 ⁇ 10 2 cells / cm 2 to 1 ⁇ 10 6 cells / cm 2 , more preferably 1 ⁇ 10 3 cells / cm 2 to 2 ⁇ 10 5 cells / cm 2 , more preferably 2 ⁇ 10 3 cells / cm 2 To 4 ⁇ 10 4 cells / cm 2 .
- the medium used in the second step is not particularly limited as long as it is as described in the above definition.
- the medium used in the second step may be a serum-containing medium or a serum-free medium, but in the present invention, a serum-free medium is preferable from the viewpoint of avoiding contamination with chemically undecided components.
- a serum-free medium for example, a medium containing 0.5% to 30% concentration range of KSR in Glasgow MEM medium
- an appropriate amount of a commercially available serum substitute such as KSR is used.
- the medium used in the second step may contain a ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor for the purpose of suppressing cell death (apoptosis).
- ROCK inhibitors include Y-27632, Fasudil, or H-1152.
- concentration of the ROCK inhibitor can be appropriately set by those skilled in the art, and can be set, for example, from a range of concentrations exhibiting ROCK inhibitory activity corresponding to about 50 nM to 200 ⁇ M of Y-27632.
- the Nodal signal transduction pathway inhibitor is not particularly limited as long as it can suppress signal transduction mediated by Nodal, and may be any of protein, nucleic acid, and low molecular weight compound. Signals mediated by Nodal are transmitted through the Nodal receptor. Nodal receptors exist as heterodimers of type I receptors (ALK (activin-receptor-like-kinase) -4, ALK-5, ALK-7) and type II receptors (ActRII).
- ALK activin-receptor-like-kinase
- ActRII type II receptors
- Nodal signal transduction pathway examples include substances that directly act on Nodal or Nodal receptor (anti-Nodal antibody, anti-Nodal receptor antibody, etc.), substances that suppress the expression of Nodal or Nodal receptor gene ( For example, antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Nodal receptor and Nodal (Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble type Nodal receptor, etc.), Nodal receptor A substance that inhibits the physiological activity resulting from signal transduction by SB-431542 (SB431542) (4- [4- (1,3-Benzodioxol-5-yl)-which inhibits the kinase activity of type I receptor by ATP competition Low molecular compounds such as 5- (pyridin-2-yl) -1H-imidazol-2-yl] benzamide), etc., but is not limited thereto. SB-431542 is a known Nodal
- the Wnt signal transduction pathway inhibitor is not particularly limited as long as it can suppress signal transduction mediated by Wnt, and may be any protein, nucleic acid, low molecular weight compound and the like. Signals mediated by Wnt are transmitted through Wnt receptors that exist as heterodimers of Frizzled (Fz) and LRP5 / 6 (low-densitylowlipoprotein receptor-related protein 5/6).
- Wnt signal transduction pathway inhibitors include substances that directly act on Wnt or Wnt receptors (anti-Wnt antibodies, anti-Wnt receptor antibodies, etc.), substances that suppress the expression of genes encoding Wnt or Wnt receptors ( For example, antisense oligonucleotides, siRNA, etc.), substances that inhibit Wnt receptor binding to Wnt (soluble Wnt receptor, dominant negative Wnt receptor, Wnt antagonist, Dkk1, Cerberus protein, etc.), depending on Wnt receptor Substances that inhibit physiological activity caused by signal transduction [CKI-7 (N- (2-Aminoethyl) -5-chloroisoquinoline-8-sulfonamide) and D4476 (4- [4- (2, 3-Dihydro-1,4-benzodioxin-6-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] benzamide), inhibiting the turnover of Axin Stabilize IWR-1-
- CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2 and the like are known Wnt signal inhibitors, and commercially available products can be appropriately obtained.
- a Wnt signal inhibitory substance CKI-7 is preferable.
- a Nodal signal transduction pathway inhibitor or a Wnt signal transduction pathway inhibitor can be used alone, but preferably both are used in combination.
- the concentration of the Nodal signal transduction pathway inhibitor and the Wnt signal transduction pathway inhibitor contained in the medium should be appropriately set by those skilled in the art within the range in which retinal pigment epithelial cells can be produced by the method of the present invention.
- its concentration is usually 0.01-50 ⁇ M, preferably 0.1-10 ⁇ M, more preferably 5 ⁇ M, and CKI- When 7 is used, the concentration is usually 0.01 to 30 ⁇ M, preferably 0.1 to 30 ⁇ M, more preferably 3 ⁇ M.
- the concentration may be constant throughout the second step or may be changed stepwise.
- the culture in the second step may be performed under any conditions of suspension culture or adhesion culture, but is preferably performed by adhesion culture.
- the incubator used for the second step adhesion culture is not particularly limited as long as cell adhesion culture is possible, but a cell adhesion incubator is preferable.
- a cell adhesion incubator an incubator that has been artificially treated for the purpose of improving the adhesion with cells can be used.
- the incubator in which the above-mentioned interior is coated with a coating agent is mentioned.
- laminin As a coating agent, laminin [including laminin ⁇ 5 ⁇ 1 ⁇ 1 (laminin 511), laminin ⁇ 1 ⁇ 1 ⁇ 1 (laminin 111) and the like and laminin fragment (laminin 511E8 etc.)], enteractin, collagen, gelatin, vitronectin (Vitronectin), synthemax (Corning) ), An extracellular matrix such as Matrigel, or a polymer such as polylysine or polyornithine, and more preferably laminin 511E8 (WO2015 / 053375).
- Laminin 511E8 can be purchased commercially (eg, iMatrix-511, Nippi).
- the incubator used for the suspension culture in the second step is not particularly limited as long as suspension culture of cells is possible, but is preferably non-cell-adhesive.
- Culture conditions such as culture temperature and CO 2 concentration in the second step can be set as appropriate.
- the culture temperature is, for example, about 30 ° C. to about 40 ° C., preferably about 37 ° C.
- the CO 2 concentration is for example about 1% to about 10%, preferably about 5%.
- the medium can be changed as appropriate.
- the method of exchanging the medium is not particularly limited, and the whole amount of the original medium may be replaced with a new medium, or only a part of the original medium may be replaced with a new medium.
- the final concentration of substances (Nodal signaling pathway inhibitor, Wnt signaling pathway inhibitor, KSR, etc.) included in the second step is calculated and then replaced.
- a new medium containing the substance at a concentration corresponding to the ratio of the medium can be prepared and replaced with the original medium.
- the final concentration of the substance included in the second step may be changed during the culture.
- the tool used for culture medium exchange operation is not specifically limited, For example, a pipetter, a micropipette, a multichannel micropipette, a continuous dispenser, etc. are mentioned.
- a pipetter a micropipette, a multichannel micropipette, a continuous dispenser, etc.
- a multichannel pipette may be used.
- the culture period of the second step is not particularly limited as long as the target retinal pigment epithelial cells can be induced. Examples of such a culture period are usually 14 to 40 days from the start of the second step.
- Retinal pigment epithelial cells can be generated in the eye.
- those skilled in the art can express cell markers (RPE65 (retinal pigment epithelial cells), Mitf (retinal pigment epithelial cells) BEST1 (retinal pigment epithelial cells), CRALBP (retinal pigment epithelial cells), etc.) It is possible to confirm the generation of retinal pigment epithelial cells based on the presence (blackish brown), tight junction between cells, and the characteristic cell shape of polygonal and paving stones. By confirming these, the culture period can be set. It is also possible to do.
- the medium is preferably replaced with a retinal pigment epithelial cell maintenance medium (hereinafter sometimes referred to as RPE maintenance medium) and further cultured.
- RPE maintenance medium retinal pigment epithelial cell maintenance medium
- melanin pigmented cells and cells having a polygonal flat shape adhering to the basement membrane can be observed more clearly.
- the culture using the RPE maintenance medium is not limited as long as colonies of retinal pigment epithelial cells are formed.
- the culture is performed for about 5 to 20 days while exchanging the whole medium at least once every 3 days.
- basal medium serum and / or Or it is composed of serum substitutes and other ingredients.
- the basal medium is not particularly limited as long as it is as described in the above definition section.
- serum serum derived from mammals such as cows and humans can be used.
- a serum substitute from the viewpoint of quality control of target cells
- B27 which is a serum substitute for nerve cell culture is particularly preferable.
- other components include L-glutamine, penicillin sodium, streptomycin sulfate and the like.
- high-purity retinal pigment epithelial cells can be obtained by performing a concentration / purification operation.
- concentration / purification method is not particularly limited as long as it is generally known as a method for concentrating / purifying cells.
- filtration eg, WO2015 / 053376
- centrifugation perfusion separation
- flow cytometry separation antibody
- a method such as trap separation using a modified carrier can be used.
- the production method of the present invention may include a step of amplifying retinal pigment epithelial cells after completion of the second step.
- retinal pigment epithelial cells can be amplified by the method described in WO2015 / 053375.
- amplification method described in WO2015 / 053375 cells with insufficient differentiation induction contained in cultured cells can be trapped, and byproducts other than retinal pigment epithelial cells can be relatively reduced.
- the amplification process can also serve as a purification process for retinal pigment epithelial cells.
- the retinal pigment epithelial cells when the retinal pigment epithelial cells are produced by suspension culture, the retinal pigment epithelial cells can be collected as a single cell and used as a suspension.
- the retinal pigment epithelial cells when retinal pigment epithelial cells are produced by adhesion culture, the retinal pigment epithelial cells adhere to each other and can take a sheet-like structure. Therefore, it is possible to produce a sheet of retinal pigment epithelial cells that can be transplanted to a patient. This sheet of retinal pigment epithelial cells is particularly useful as a cell population used as a cell transplantation therapeutic agent for treating retinal diseases.
- the retinal pigment epithelial cells produced by adhesion culture are detached by the above-described method, and the retinal pigment epithelial cells are collected as independent single cells, and these are recovered with a physiological aqueous solvent (saline, buffer, It can also be prepared as a suspension by suspending it in a serum medium or the like.
- a physiological aqueous solvent saline, buffer, It can also be prepared as a suspension by suspending it in a serum medium or the like.
- Retinal pigment epithelial cells produced by the production method of the present invention are used as healthy and disease model cells as therapeutic agents for retinal diseases and other complications such as diabetes, or preventive agents thereof. It can be used for screening / evaluation of drug efficacy, safety test for chemical substances, stress test, toxicity test, side effect test, infection / contamination test. On the other hand, it can also be used for toxicity studies such as phototoxicity and retinal excitotoxicity specific to retinal cells, and toxicity tests.
- the evaluation methods include stimulation and toxicity tests such as apoptosis evaluation, as well as tests to evaluate the effect on normal differentiation from progenitor cells to retinal pigment epithelial cells and photoreceptor cells (RT-PCR for various gene markers, ELISA for cytokines) Analysis of expressed protein, phagocytic ability test), toxicity test such as phototoxicity, retinal potential and transepithelial electrical resistance to visual function, and cytotoxicity test caused by autoimmune reaction.
- RT-PCR for various gene markers
- ELISA cytokines
- toxicity test such as phototoxicity, retinal potential and transepithelial electrical resistance to visual function
- cytotoxicity test caused by autoimmune reaction cytotoxicity test caused by autoimmune reaction.
- cell materials for these tests not only retinal pigment epithelial cells but also progenitor cells thereof can be used. For example, cells seeded and adhered, cell suspensions, sheets or molded articles thereof Can be provided. This can be used as an extrapolation test for human and animal studies.
- composition provides a pharmaceutical composition comprising an effective amount of retinal pigment epithelial cells produced by the production method of the present invention.
- the pharmaceutical composition contains an effective amount of retinal pigment epithelial cells produced by the production method of the present invention and a pharmaceutically acceptable carrier.
- a physiological aqueous solvent physiological saline, buffer solution, serum-free medium, etc.
- a preservative, a stabilizer, a reducing agent, an isotonic agent, and the like that are usually used may be added to the medicine containing the tissue or cells to be transplanted.
- the pharmaceutical composition of the present invention can be produced as a suspension by suspending the retinal pigment epithelial cells produced by the production method of the present invention with an appropriate physiological aqueous solvent.
- a cryopreservation agent may be added, cryopreserved with liquid nitrogen, etc., thawed at the time of use, washed with a buffer solution, and used for transplantation medicine.
- the retinal pigment epithelial cells obtained by the production method of the present invention can be cut into appropriate sizes using tweezers or the like to form a sheet.
- the cell obtained by the manufacturing method of this invention can also be shape
- the pharmaceutical composition of the present invention is useful as a therapeutic agent for diseases based on (caused by) retinal pigment epithelial cell disorders.
- Retinal pigment therapeutic agent and therapeutic method Retinal pigment epithelial cells produced by the production method of the present invention (including retinal pigment epithelial cells that have undergone the above-described concentration and amplification operations, etc.) are transplanted into a living body in the form of a suspension or sheet. It can be used as a cell transplantation therapeutic agent for treating retinal diseases.
- the present invention also provides a treatment method comprising administering the therapeutic agent to a patient.
- the retinal disease is an ophthalmic disease related to the retina, and includes complications due to other diseases such as diabetes.
- retinal diseases in the present invention include diseases based on retinal pigment epithelial cell disorders, such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy or retinal detachment. That is, retinal pigment epithelial cells produced by the production method of the present invention can be supplemented to the damaged site of retinal pigment epithelial cells in a patient.
- diseases based on retinal pigment epithelial cell disorders such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy or retinal detachment. That is, retinal pigment epithelial cells produced by the production method of the present invention can be supplemented to the damaged site of retinal pigment epithelial cells in a patient.
- pluripotent stem cells eg, induced pluripotent stem cells
- pluripotent stem cells eg, induced pluripotent stem cells
- a pluripotent stem cell in the method of the present invention, as a pluripotent stem cell, a pluripotent stem cell established from a somatic cell of another person whose immunity is compatible with the recipient, an allo retinal pigment epithelial cell, or a tissue containing the same And is transplanted to the recipient.
- a pluripotent stem cell established from a recipient's somatic cell eg, induced pluripotent stem cell
- an immunological self retinal pigment epithelial cell or a tissue containing the same is produced for the recipient, This is transplanted to the recipient.
- human dermal fibroblast-derived iPS cells (201B7, Kyoto University), and human peripheral blood-derived mononuclear cell-derived iPS established by Kyoto University from Cellular Technology Limited Limited ePMBC (registered trademark) Cells (1231A3, Ff-I01, QHJI01) were used.
- Example 1 Production of highly efficient retinal pigment epithelial cells using human iPS cells including a MEK inhibitor treatment step Undifferentiated maintenance culture of human iPS cells (201B7 strain and 1231A3 strain) under feeder-free conditions, According to the method described in “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594”.
- As the medium “StemFit (registered trademark)” AK03 medium (Ajinomoto) (hereinafter referred to as AK03 medium) or Essential 8 medium (Life Technologies) was used.
- Production of retinal pigment epithelium (RPE) cells including the MEK inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After single dispersion, 1.2 x 10 4 cells per well were seeded in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) The drug)]-containing AK03 medium or Essential 8 medium was cultured under conditions of 37 ° C. and 5% CO 2 .
- PD0325901 (SIGMA) as a MEK inhibitor was added to AK03 medium to a final concentration of 1 ⁇ M or Essential 8 medium to a final concentration of 0.03 ⁇ M (start of the first step) and exposed for 6 days (first stage) Process end).
- the cells were then treated with 0.5 x TrypLE select, detached with a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) per well,
- iMatrix-511 0.5 ⁇ g / cm 2
- % KSR, Y-27632 (Final concentration 10 ⁇ M), basal medium supplemented with SB-431542 (final concentration 5 ⁇ M), CKI-7 (final concentration 3 ⁇ M), basal supplemented with only 10% KSR from day 14 to 30 of culture Medium, RPE maintenance medium [67% DMEM low glucose (SIGMA), 29% F12 (SIGMA), 1.9% B-27 supplement (Life Technologies), 1.9 mM L-glutamine, 96 U / ml] Penicillin-96 ⁇ g / ml streptomycin] was used. The whole medium was changed every day.
- RPE maintenance medium [67% DMEM low glucose (SIGMA), 29% F12 (SIGMA), 1.9% B-27 supplement (Life Technologies), 1.9 mM L-glutamine, 96 U / ml] Penicillin-96 ⁇ g / ml streptomycin] was used. The whole medium was changed every day.
- Comparative Example 1 Manufacture of retinal pigment epithelial cells using human iPS cells without the MEK inhibitor treatment step Undifferentiated maintenance culture of human iPS cells (201B7 and 1231A3 strains) under feeder-free conditions , M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594 ”.
- As the medium “StemFit (registered trademark)” AK03 medium (Ajinomoto) (hereinafter, AK03 medium) or Essential 8 medium (Life Technologies) was used.
- Production of retinal pigment epithelium (RPE) cells not including the MEK inhibitor treatment step was performed as follows.
- IPS cells that had been cultured without maintenance were treated with 0.5 x TrypLE select, detached with a cell scraper, dispersed by pipetting, and coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ).
- iMatrix-511 Nippi
- a 6-well culture plate 2.0 ⁇ 10 5 cells were seeded per well when using AK03 medium, and 5.0 ⁇ 10 5 cells were seeded when using Essential 8 medium, and cultured under conditions of 37 ° C. and 5% CO 2 .
- RPE maintenance medium [67% DMEM low glucose (SIGMA), 29% F12 (SIGMA), 1.9% B-27 supplement (Life Technologies), 1.9 mM L-glutamine, 96 U / ml penicillin-96 ⁇ g / ml streptomycin].
- SIGMA DMEM low glucose
- SIGMA 29% F12
- B-27 supplement Life Technologies
- 1.9 mM L-glutamine 96 U / ml penicillin-96 ⁇ g / ml streptomycin
- Example 2 Production of highly efficient retinal pigment epithelial cells using human iPS cells including FGF receptor inhibitor treatment step
- Undifferentiated maintenance culture of human iPS cells (201B7 and 1231A3 strains) under feeder-free conditions was performed according to the method described in “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594”.
- As the medium “StemFit (registered trademark)” AK03 medium (Ajinomoto) (hereinafter referred to as AK03 medium) or Essential 8 medium (Life Technologies) was used.
- Production of retinal pigment epithelium (RPE) cells including the FGF receptor inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After single dispersion, 1.2 x 10 4 cells per well were seeded in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) The drug)]-containing AK03 medium or Essential 8 medium was cultured under conditions of 37 ° C. and 5% CO 2 .
- StemSure hPSC Medium ⁇ (hereinafter referred to as StemSure hPSC Medium ⁇ w) without addition of bFGF was used to examine the effects of FGF receptor inhibitors in a medium that does not contain undifferentiated maintenance factor (bFGF). / o bFGF) was used.
- PD173074 SIGMA as an FGF receptor inhibitor was added to StemSure hPSC Medium ⁇ w / o bFGF to a final concentration of 100 nM (start of the first step) and exposed for 6 days (end of the first step) .
- the cells were then treated with 0.5 x TrypLE select, detached with a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) per well, When using AK03 medium or StemSure hPSC Medium ⁇ w / o bFGF, 2.0 x 10 5 cells were seeded, and when using Essential 8 medium, 5.0 x 10 5 cells were seeded and cultured under conditions of 37 ° C and 5% CO 2 (second step) start).
- Y-27632 (Wako Pure Chemical Industries) (final concentration 10 ⁇ M) as a ROCK inhibitor
- SB-431542 (Wako Pure Chemical Industries) (final concentration 5 ⁇ M) as a Nodal signaling pathway inhibitor
- Wnt signaling AK03 medium a Nodal signaling pathway inhibitor
- Essential8 medium a Nodal signaling pathway inhibitor
- StemSure hPSC Medium ⁇ w / o bFGF supplemented with CKI-7 (SIGMA) (final concentration 3 ⁇ M) as a pathway inhibitor was used.
- % KSR (Life Technologies), Y-27632 (final concentration 10 ⁇ M), SB-431542 (final concentration 5 ⁇ M), basal medium supplemented with CKI-7 (final concentration 3 ⁇ M)
- GMEM medium SIGMA
- 0.1 mM MEM non-essential amino acid solution Life Technologies
- 1 mM sodium pyruvate SIGMA
- 0.1 mM 2-mercaptoethanol Wako Pure Chemical Industries
- 2 mM L-glutamine SIGMA
- 100 U / ml penicillin-100 ⁇ g / ml Streptomycin (Life Technologies) from 6th to 9th culture, 15% KSR, Y-27632 (final concentration 10 ⁇ M), SB-431542 (final concentration 5 ⁇ M), CKI-7 (final concentration 3 ⁇ M)
- the whole medium was changed every day.
- a black brown color was observed in the 201B7 strain and / or the 1231A3 strain.
- a wide range of cell populations was observed (Fig. 4).
- the cells showed typical features of RPE cells such as dark brown, polygonal, and cobblestone morphology ( Figure 5).
- Comparative Example 2 Production of retinal pigment epithelial cells using human iPS cells that do not include a FGF receptor inhibitor treatment step Undifferentiated maintenance culture of human iPS cells (201B7 strain and 1231A3 strain) under feeder-free conditions, According to the method described in “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594”. “StemFit (registered trademark)” AK03 medium (Ajinomoto) (hereinafter referred to as AK03 medium) or Essential 8 medium (Life Technologies) was used. Production of retinal pigment epithelium (RPE) cells not including the FGF receptor inhibitor treatment step was performed as follows.
- IPS cells that have been cultured undifferentiated or iPS cells that have been cultured for 6 days in StemSure hPSC Medium ⁇ w / o bFGF (Wako Pure Chemical Industries, Ltd.) are treated with 0.5 x TrypLE select, detached using a cell scraper, and removed. After monodispersion by petting, per well of 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), AK03 medium or 2.0 x when using StemSure hPSC Medium ⁇ w / o bFGF When using 10 5 cells and Essential 8 medium, 5.0 ⁇ 10 5 cells were seeded and cultured under conditions of 37 ° C.
- % KSR (Life Technologies), Y-27632 (final concentration 10 ⁇ M), SB-431542 (final concentration 5 ⁇ M), basal medium supplemented with CKI-7 (final concentration 3 ⁇ M)
- GMEM medium SIGMA
- 0.1 mM MEM non-essential amino acid solution Life Technologies
- 1 mM sodium pyruvate SIGMA
- 0.1 mM 2-mercaptoethanol Wako Pure Chemical Industries
- 2 mM L-glutamine SIGMA
- 100 U / ml penicillin-100 ⁇ g / ml Streptomycin (Life Technologies) from 6th to 9th culture, 15% KSR, Y-27632 (final concentration 10 ⁇ M), SB-431542 (final concentration 5 ⁇ M), CKI-7 (final concentration 3 ⁇ M)
- Example 3 High-efficiency retina using human iPS cells, comprising a combination treatment step of a MEK inhibitor and / or FGF receptor inhibitor and various inhibitors, signal transduction pathway inhibitors or signal transduction pathway agonists Production of pigment epithelial cells Undifferentiated maintenance culture of human iPS cells (201B7 and 1231A3 strains) under feeder-free conditions is described in “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594”. In accordance with the method described in 1. As the medium, “StemFit (registered trademark)” AK03 medium (Ajinomoto) (hereinafter, AK03 medium) was used.
- RPE retinal pigment epithelium
- IPS cells that had been cultured without maintenance were treated with 0.5 x TrypLE select, detached with a cell scraper, dispersed by pipetting, and coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ).
- Nippi iMatrix-511
- a 6-well culture plate was seeded with 1.2 ⁇ 10 4 cells per well and cultured in an AK03 medium containing a ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure Chemical Industries)] at 37 ° C. and 5% CO 2 .
- PD0325901 (final concentration 1 ⁇ M) as MEK inhibitor
- PD173074 (SIGMA) (final concentration 100 nM) as FGF receptor inhibitor
- LDN193189 (STEMGENT) (final concentration 100) as BMP receptor inhibitor nM
- SAG Enzo Life Sciences
- Go6983 (SIGMA) (final concentration 2 ⁇ M) as a PKC inhibitor
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) at a rate of 2.0 per well.
- x 10 5 cells were seeded and cultured under conditions of 37 ° C. and 5% CO 2 (start of the second step).
- Example 1 including the MEK inhibitor treatment step, Comparative Example 1 not including the MEK inhibitor treatment step, Example 2 including the FGF receptor inhibitor treatment step, and no FGF receptor inhibitor treatment step The experiment was also performed under the conditions of Comparative Example 2. As a result of observing the culture plate from the 38th day to the 47th day of the culture, under the conditions of Comparative Example 1 and Comparative Example 2, the appearance of cells exhibiting typical characteristics of RPE cells such as dark brown, polygonal, and cobblestone morphology was slight. ( Figure 7, no treatment).
- Manufacturing conditions including combination treatment with substances, signal transduction pathway inhibitors or signal transduction pathway agonists can confirm the widespread appearance of RPE cells exhibiting typical RPE cell characteristics such as dark brown, polygonal, and cobblestone morphology.
- Figure 7 When the ratio of RPE cells occupying the whole well was visually determined and divided into 6 stages from 0 to 5 according to the ratio (Fig. 8A), all the compound combination treatment conditions shown in Figs. The appearance of a higher proportion of RPE cells was also confirmed (FIGS. 8B and 8C).
- Example 4 Production of highly efficient retinal pigment epithelial cells using human iPS cells Ff-I01 and QHJI01 including a MEK inhibitor or FGF receptor inhibitor treatment step
- Human iPS cells Ff-I01 strain and QHJI01 strain
- the undifferentiated maintenance culture under feeder-free conditions was performed according to the method described in “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594”.
- As the medium “StemFit (registered trademark)” AK03N medium (Ajinomoto) (hereinafter referred to as “AK03N medium”) was used.
- Production of retinal pigment epithelium (RPE) cells including a MEK inhibitor or FGF receptor inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After monodispersion, inoculate 2.0 x 10 4 cells per well in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and add ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing [medicine)], the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- PD0325901 (SIGMA) (final concentration 1 ⁇ M) as a MEK inhibitor or PD173074 (SIGMA) (final concentration 100 nM) as an FGF receptor inhibitor was added to the AK03N medium (start of the first step), 6 Exposure for 1 day (end of the first step).
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) at a rate of 2.0 per well.
- x 10 5 cells were seeded and cultured under conditions of 37 ° C. and 5% CO 2 (start of the second step).
- Comparative Example 1 that does not include the MEK inhibitor treatment step (however, AK03 medium is changed to AK03N medium) and Comparative Example 2 that does not include the FGF receptor inhibitor treatment step (however, AK03 medium is AK03N medium)
- Comparative Example 2 that does not include the FGF receptor inhibitor treatment step (however, AK03 medium is AK03N medium)
- the experiment was also carried out.
- the appearance of cells having typical characteristics of RPE cells such as blackish brown, polygonal, and cobblestone morphology was hardly seen ( Figure 9, no processing).
- a MEK inhibitor and an FGF receptor inhibitor when exposed to a MEK inhibitor and an FGF receptor inhibitor, a wide range of black-brown cell populations could be confirmed in both Ff-I01 and QHJI01 strains (FIG. 9, MEKi, FGFRi).
- Example 5 Examination of exposure days of each inhibitory substance in the MEK inhibitory substance or FGF receptor inhibitory substance treatment step Undifferentiated maintenance culture of human iPS cells (QHJI01 strain) under feeder-free conditions is described in “Nakagawa, M. et al. al., Sci. Rep. 2014 Jan 8; 4: 3594 ”.
- As the medium “StemFit (registered trademark)” AK03N medium (Ajinomoto) (hereinafter referred to as “AK03N medium”) was used.
- Production of retinal pigment epithelium (RPE) cells including a MEK inhibitor or FGF receptor inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After monodispersion, inoculate 2.0 x 10 4 cells per well in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and add ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) Drug)] In an AK03N-containing medium, the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- PD0325901 SIGMA
- MEK inhibitor final concentration 1 ⁇ M
- PD173074 SIGMA
- FGF receptor inhibitor final 100 nM
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) at a rate of 2.0 per well.
- x 10 5 cells were seeded and cultured under conditions of 37 ° C. and 5% CO 2 (start of the second step). From day 1 to day 4 of culture, 20% KSR (Life Technologies), Y-27632 (final concentration 10 ⁇ M), SB-431542 (Wako Pure Chemical Industries) (final concentration 5 ⁇ M) as a Nodal signaling pathway inhibitor, Basal medium supplemented with CKI-7 (SIGMA) (final concentration 3 ⁇ M) as a Wnt signaling pathway inhibitor [GMEM medium (SIGMA), 0.1 mM MEM non-essential amino acid solution (Life Technologies), 1 mM sodium pyruvate (SIGMA) ), 0.1 mM 2-mercaptoethanol (Wako Pure Chemicals), 2 mM L-glutamine (SIGMA), 100 U / ml penicillin-100 ⁇ g / ml streptomycin (Life Technologies)], Basal medium supplemented with 15% KSR, Y-27632 (final concentration 10 ⁇ M),
- Example 6 Examination of exposure period of MEK inhibitor in MEK inhibitor treatment process Undifferentiated maintenance culture of human iPS cells (1231A3 strain) under feeder-free conditions is described in “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594 ”. As the medium, “StemFit (registered trademark)” AK03 medium (Ajinomoto) (hereinafter, AK03 medium) was used. Production of retinal pigment epithelium (RPE) cells including the MEK inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After single dispersion, 1.2 x 10 4 cells per well were seeded in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) In the AK03 medium containing the drug), the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- iMatrix-511 0.5 ⁇ g / cm 2
- ROCK inhibitor 10 ⁇ M Y-27632 (Wako Pure) In the AK03 medium containing the drug
- PD0325901 SIGMA (final concentration 1 ⁇ M) was added to the AK03 medium as the MEK inhibitor (starting the first step) and exposed for 6 days, 3 days, or 1 day. (End of the first step).
- Cells exposed to MEK inhibitors for 6 days are treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ) Inoculate 0.8 x 10 4 cells per well in a 6-well culture plate, and use AK03 medium containing Y-27632 (final concentration 10 ⁇ M) and PD0325901 (final concentration 1 ⁇ M) at 37 ° C and 5% CO 2 By further culturing for 7 days, the cells were exposed to a MEK inhibitor for 13 days (end of the first step).
- iMatrix-511 Nippi
- AK03 medium containing Y-27632 final concentration 10 ⁇ M
- PD0325901 final concentration 1 ⁇ M
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ). x 10 6 cells were seeded and cultured under conditions of 37 ° C. and 5% CO 2 (start of the second step).
- Example 7 Gene expression at the end of the MEK inhibitor or FGF receptor inhibitor treatment step
- Undifferentiated maintenance culture of human iPS cells (Ff-I01 strain and QHJI01 strain) under feeder-free conditions is described in “Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594 ”.
- As the medium “StemFit (registered trademark)” AK03N medium (Ajinomoto) (hereinafter referred to as “AK03N medium”) was used.
- Production of retinal pigment epithelium (RPE) cells including a MEK inhibitor or FGF receptor inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After monodispersion, inoculate 2.0 x 10 4 cells per well in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and add ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing [medicine)], the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- PD0325901 SIGMA
- PD173074 SIGMA
- FGF receptor inhibitor FGF receptor inhibitor
- AK03N medium first (Step started), exposed for 6 days or 3 days (end of first step).
- cells are treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then used for RNA extraction as a microarray sample except for the cells that are transferred to the second step.
- basal medium containing 20% KSR, Y-27632, SB-431542, and CKI-7)
- basal medium containing 20% KSR, Y-27632, SB-431542, and CKI-7
- Basal medium supplemented with Y-27632 (final concentration 10 ⁇ M), SB-431542 (final concentration 5 ⁇ M), CKI-7 (final concentration 3 ⁇ M), 15% KSR, Y on the 5th to 8th day of culture -27632 (final concentration 10 ⁇ M), SB-431542 (final concentration 5 ⁇ M)
- basal medium supplemented with CKI-7 final concentration 3 ⁇ M
- 10% KSR Y- Basal medium supplemented with 27632 (final concentration 10 ⁇ M), SB-431542 (final concentration 5 ⁇ M), CKI-7 (final concentration 3 ⁇ M)
- only 10% KSR was added on days 13 to 30 of culture RPE maintenance medium was used after the basal medium and culture day 31.
- the whole medium was changed every day.
- the microarray specimen was collected and the RPE cells were produced under the conditions of Comparative Example 1 not including the MEK inhibitor treatment step (AK03 medium was changed to AK03N medium).
- RNeasy Mini Kit QIAGEN
- microarray analysis was performed using GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix).
- the microarray analysis used the commissioned analysis of Kurashiki Textile Co., Ltd. Based on the observation image of the culture plate on the 43rd day of culture, the result of visual determination of the ratio of RPE cells occupying the whole well according to FIG.
- Example 8 Examination of various inhibitor concentrations in the MEK inhibitor or FGF receptor inhibitor treatment step Undifferentiated maintenance culture of human iPS cells (QHJI01 strain) under feeder-free conditions is described in “Nakagawa, M. et. Al. ., Sci. Rep. 2014 Jan 8; 4: 3594 ”. As the medium, “StemFit (registered trademark)” AK03N medium (Ajinomoto) (hereinafter referred to as “AK03N medium”) was used. Production of retinal pigment epithelium (RPE) cells including a MEK inhibitor or FGF receptor inhibitor treatment step was performed as follows.
- RPE retinal pigment epithelium
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After monodispersion, inoculate 2.0 x 10 4 cells per well in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and add ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing [medicine)], the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- PD0325901 (SIGMA) as a MEK inhibitor at a final concentration of 0.25 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 2 ⁇ M, or 4 ⁇ M
- PD173074 (SIGMA) as an FGF receptor inhibitor at a final concentration of 25 nM, 50 nM
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) at a rate of 2.0 per well.
- x 10 5 cells were seeded and cultured under conditions of 37 ° C. and 5% CO 2 (start of the second step).
- Example 9 Examination of the number of seeded cells during the transition from the first step to the second step
- the undifferentiated maintenance culture of human iPS cells (QHJI01 strain) under feeder-free conditions was performed according to “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594 ”.
- As the medium “StemFit (registered trademark)” AK03N medium (Ajinomoto) (hereinafter referred to as “AK03N medium”) was used.
- Production of retinal pigment epithelium (RPE) cells including a MEK inhibitor or FGF receptor inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After monodispersion, inoculate 2.0 x 10 4 cells per well in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and add ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing the drug), the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- iMatrix-511 0.5 ⁇ g / cm 2
- ROCK inhibitor 10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing the drug
- PD0325901 (SIGMA) (final concentration 1 ⁇ M) as a MEK inhibitor or PD173074 (SIGMA) (final concentration 100 nM) as an FGF receptor inhibitor was added to the AK03N medium (start of the first step), 6 Exposure for 1 day (end of the first step).
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ).
- Example 10 Examination of MEK inhibitors PD184352, U0126, TAK-7331, and AZD-8330 in the first step Undifferentiated maintenance culture of human iPS cells (QHJI01 strain and 1231A3 strain) under feeder-free conditions was performed according to “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594 ”. As the medium, “StemFit (registered trademark)” AK03N medium (Ajinomoto) (hereinafter referred to as “AK03N medium”) was used. Production of retinal pigment epithelium (RPE) cells including the MEK inhibitor treatment step was performed as follows.
- RPE retinal pigment epithelium
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After monodispersion, inoculate 2.0 x 10 4 cells per well in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and add ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing the drug), the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- iMatrix-511 0.5 ⁇ g / cm 2
- ROCK inhibitor 10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing the drug
- PD0325901 SIGMA
- PD184352 SIGMA
- U0126 SIGMA
- TAK as MEK inhibitors -7331 Selleck
- AZD-8330 Selleck
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) at a rate of 2.0 per well.
- x 10 5 cells were seeded and cultured under conditions of 37 ° C. and 5% CO 2 (start of the second step).
- Example 11 Examination of FGF receptor inhibitor SU5402 in the first step Undifferentiated maintenance culture of human iPS cells (QHJI01 strain and 1231A3 strain) under feeder-free conditions is described in “Nakagawa, M. et. Al., Sci”. Rep. 2014 Jan 8; 4: 3594 ”. As the medium, “StemFit (registered trademark)” AK03N medium (Ajinomoto) (hereinafter referred to as “AK03N medium”) was used. Production of retinal pigment epithelium (RPE) cells including the FGF receptor inhibitor treatment step was performed as follows.
- RPE retinal pigment epithelium
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After monodispersion, inoculate 2.0 x 10 4 cells per well in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and add ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing the drug), the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- iMatrix-511 0.5 ⁇ g / cm 2
- ROCK inhibitor 10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing the drug
- PD173074 (SIGMA) (final concentration 100 nM) or SU5402 (SIGMA) (final concentrations 5 ⁇ M, 10 ⁇ M, 20 ⁇ M) is added to the AK03N medium as the FGF receptor inhibitor (start of the first step) , Exposed for 6 days (end of the first step).
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) at a rate of 2.0 per well.
- x 10 5 cells were seeded and cultured under conditions of 37 ° C.
- Example 12 Examination of differentiation induction effect by single exposure of Nodal signaling pathway inhibitor and Wnt signaling pathway inhibitor in the second step Human iPS cells (QHJI01 strain) undifferentiated maintenance culture under feeder-free conditions, According to the method described in “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594”.
- As the medium “StemFit (registered trademark)” AK03N medium (Ajinomoto) (hereinafter referred to as “AK03N medium”) was used.
- Production of retinal pigment epithelium (RPE) cells including the MEK inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After monodispersion, inoculate 2.0 x 10 4 cells per well in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and add ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing the drug), the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- iMatrix-511 0.5 ⁇ g / cm 2
- ROCK inhibitor 10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing the drug
- PD0325901 SIGMA (final concentration 1 ⁇ M) was added to the AK03N medium as a MEK inhibitor (first step started) and exposed for 6 days (end of the first step).
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 12-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ).
- x 10 5 cells were seeded and cultured under conditions of 37 ° C. and 5% CO 2 (start of the second step). In the second step, the following three types of media were used.
- Example 13 Relationship Between Area Ratio of Black- Brown Cells in Whole Well and Expression of Retinal Pigment Epithelial Cell Marker Gene
- Undifferentiated maintenance culture of human iPS cells (201B7 strain) under feeder-free conditions is described in “Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594 ”.
- As the medium “StemFit (registered trademark)” AK03 medium (Ajinomoto) (hereinafter, AK03 medium) was used.
- Production of retinal pigment epithelium (RPE) cells including a MEK inhibitor and BMP receptor inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After single dispersion, 1.2 x 10 4 cells per well were seeded in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) Drug)] was cultured in AK03 medium containing 37 ° C. and 5% CO 2 .
- iMatrix-511 0.5 ⁇ g / cm 2
- ROCK inhibitor 10 ⁇ M Y-27632 (Wako Pure) Drug
- PD0325901 SIGMA (final concentration 1 ⁇ M) is added to the AK03 medium as the MEK inhibitor the day after sowing (start of the first step) 6 days after sowing, LDN193189 (STEMGENT) (final concentration 100 nM) as a BMP receptor inhibitor was added to the AK03 medium to expose the MEK inhibitor and BMP receptor inhibitor for 6 days and 1 day, respectively (1st Process end).
- PD0325901 final concentration 1 ⁇ M
- LDN193189 final concentration 100 nM
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) at a rate of 2.0 per well.
- x 10 5 cells were seeded and cultured under conditions of 37 ° C. and 5% CO 2 (start of the second step).
- the expression level of BEST1, MITF, and RAX in each sample was corrected with the expression level of GAPDH, and expressed as a relative value when the expression level of iPS cells (undifferentiated) cultured under undifferentiated maintenance culture conditions was 1. .
- the proportion of RPE cells in the whole well was visually determined according to FIG. 8A. As a result, “1” was given for no treatment, “3” was for MEK inhibitor 6 days + BMP receptor inhibitor 1 day, and “5” was MEK inhibitor 6 days + BMP receptor inhibitor 6 days ( Figure) 18 top, cell photo).
- Example 14 Confirmation of expression of marker gene of RPE cell produced by a production method including a MEK inhibitor or FGF receptor inhibitor treatment step Undifferentiated maintenance culture under feeder-free conditions of human iPS cells (1231A3 strain) According to the method described in “Nakagawa, M. et. Al., Sci. Rep. 2014 Jan 8; 4: 3594”.
- As the medium “StemFit (registered trademark)” AK03N medium (Ajinomoto) (hereinafter referred to as “AK03N medium”) was used.
- Production of retinal pigment epithelium (RPE) cells including a MEK inhibitor or FGF receptor inhibitor treatment step was performed as follows.
- IPS cells that have been cultured without maintenance are treated with 0.5 x TrypLE select (equal mix of TrypLE select (Life Technologies) and 0.5 mM EDTA / PBS (-)), then detached using a cell scraper and pipetted. After monodispersion, inoculate 2.0 x 10 4 cells per well in a 6-well culture plate coated with iMatrix-511 (Nippi) (0.5 ⁇ g / cm 2 ), and add ROCK inhibitor [10 ⁇ M Y-27632 (Wako Pure) In the AK03N medium containing [medicine)], the cells were cultured under conditions of 37 ° C. and 5% CO 2 .
- PD0325901 (SIGMA) (final concentration 1 ⁇ M) as a MEK inhibitor or PD173074 (SIGMA) (final concentration 100 nM) as an FGF receptor inhibitor was added to the AK03N medium (start of the first step), 6 Exposure for 1 day (end of the first step).
- the cells were then treated with 0.5 x TrypLE select, detached using a cell scraper, monodispersed by pipetting, and then applied to a 6-well culture plate coated with iMatrix-511 (0.5 ⁇ g / cm 2 ) at a rate of 2.0 per well.
- x 10 5 cells were seeded and cultured under conditions of 37 ° C. and 5% CO 2 (start of the second step).
- RNA was extracted After observation on the 43rd day of culture, cells were collected, RNA was extracted, and RT-PCR was performed. RNeasy Micro Kit (QIAGEN) was used for RNA extraction, Oligo (dT) 12-18 Primer (Invitrogen), SuperScript III Reverse Transcriptase (Invitrogen) was used for reverse transcription, and Blend Taq-Plus- (TOYOBO) was used for PCR. .
- the primer sequences of RPE65, BEST1, CRLBP, and GAPDH are as follows.
- RPE65-F TCCCCAATACAACTGCCACT (SEQ ID NO: 1), RPE65-R: CCTTGGCATTCAGAATCAGG (SEQ ID NO: 2), BEST1-F: TAGAACCATCAGCGCCGTC (SEQ ID NO: 3), BEST1-R: TGAGTGTAGTGTGTATGTTGG (SEQ ID NO: 4), CRALBP-F: GAGGGAGCGA SEQ ID NO: 5), CRALBP-R: TGCAGAAGCCATTGATTTGA (SEQ ID NO: 6), GAPDH-F: ACCACAGTCCATGCCATCAC (SEQ ID NO: 7), GAPDH-R: TCCACCACCCTGTTGCTGTA (SEQ ID NO: 8).
- the number of PCR reaction cycles was 30 for RPE65, BEST1, and GAPDH, and 35 for CRALBP.
- the PCR product was detected by agarose gel electrophoresis as a single band around 369 bp for RPE65, 261 bp for BEST1, 341 bp for CRALBP, and 452 bp for GAPDH.
- Primary human RPE hRPE was used as a positive control, and iPS cells (undifferentiated iPSC) cultured under undifferentiated maintenance culture conditions were used as a negative control.
- the production method of the present invention makes it possible to produce retinal pigment epithelial cells from pluripotent stem cells with high efficiency.
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Abstract
Description
すなわち、本発明は以下に関する。
(1)多能性幹細胞を、FGF受容体阻害物質及び/又はMEK阻害物質を含む培地で、30日を超えない期間培養する第一工程、及び
(2)第一工程で得られた細胞を、Nodalシグナル伝達経路阻害物質及び/又はWntシグナル伝達経路阻害物質の存在下において培養し、網膜色素上皮細胞を形成させる第二工程。
[2]第一工程が、無血清条件下で行われる、上記[1]に記載の製造方法。
[3]第一工程が、フィーダー細胞非存在下で行われる、上記[1]又は[2]に記載の製造方法。
[4]第一工程における培地が、さらに未分化維持因子を含む、上記[1]~[3]のいずれかに記載の製造方法。
[5]未分化維持因子が、FGFシグナル伝達経路作用物質である、上記[4]に記載の製造方法。
[6]FGFシグナル伝達経路作用物質が、bFGFである、上記[5]に記載の製造方法。
[7]FGF受容体阻害物質が、PD173074及びSU5402からなる群から選択される少なくとも1種である、上記[1]~[6]のいずれかに記載の製造方法。
[8]MEK阻害物質が、PD0325901、PD184352、U0126、TAK-733、及びAZD-8330からなる群から選択される少なくとも1種である、上記[1]~[7]のいずれかに記載の製造方法。
[9]Nodalシグナル伝達経路阻害物質が、ALK4,5,又は7阻害物質である、上記[1]~[8]のいずれかに記載の製造方法。
[10]ALK4,5,又は7阻害物質が、SB431542である、上記[9]に記載の製造方法。
[11]Wntシグナル伝達経路阻害物質が、CKI-7である上記[1]~[10]のいずれかに記載の製造方法。
[12]多能性幹細胞が、霊長類多能性幹細胞である上記[1]~[11]のいずれかに記載の製造方法。
[13]多能性幹細胞が、ヒト多能性幹細胞である上記[1]~[12]のいずれかに記載の製造方法。
[14]第一工程における培地が、さらにBMP受容体阻害物質を含む、上記[1]~[13]のいずれかに記載の製造方法。
[15]BMP受容体阻害物質が、ALK2/3阻害物質である上記[14]に記載の製造方法。
[16]ALK2/3阻害物質が、LDN193189である上記[15]に記載の製造方法。
[17]第一工程における培地が、さらにソニック・ヘッジホッグシグナル伝達経路作用物質を含む上記[1]~[16]のいずれかに記載の製造方法。
[18]ソニック・ヘッジホッグシグナル伝達経路作用物質が、SAGである上記[17]に記載の製造方法。
[19]第一工程における培地が、さらにPKC阻害物質を含む上記[1]~[18]のいずれかに記載の製造方法。
[20]PKC阻害物質が、Go6983である上記[19]に記載の製造方法。
[21’]第一工程において、培養期間が、眼形成転写因子の少なくとも1つの遺伝子発現を誘導するのに十分でありかつ30日を超えない期間である、上記[1]~[20]のいずれかに記載の製造方法。
[22’]第一工程において、培養期間が、PAX6、LHX2及びSIX3の少なくとも1つの遺伝子発現を誘導するのに十分でありかつ30日を超えない期間である、上記[1]~[20]のいずれかに記載の製造方法。
[21]第一工程において、培養期間が2日間~13日間である、上記[1]~[20]、[21’]及び[22’]のいずれかに記載の製造方法。
[22]第一工程において、培養期間が4日間~6日間である、上記[1]~[21]、[21’]及び[22’]のいずれかに記載の製造方法。
[23]上記[1]~[22]、[21’]及び[22’]のいずれかに記載の方法により製造される網膜色素上皮細胞を含有してなる、被験物質の毒性・薬効評価用試薬。
[24]上記[1]~[22]、[21’]及び[22’]のいずれかに記載の方法により製造される網膜色素上皮細胞に被験物質を接触させ、該物質が該細胞に及ぼす影響を検定することを含む、該物質の毒性・薬効評価方法。
[25]上記[1]~[22]、[21’]及び[22’]のいずれかに記載の方法により製造される網膜色素上皮細胞を含有してなる、網膜色素上皮細胞の障害に基づく疾患の治療薬。
[26]上記[1]~[22]、[21’]及び[22’]のいずれかに記載の方法により製造される網膜色素上皮細胞の有効量を、移植を必要とする対象に移植することを含む、網膜色素上皮細胞の障害に基づく疾患の治療方法。
[27]網膜色素上皮細胞の障害に基づく疾患の治療における使用のための、上記[1]~[22]、[21’]及び[22’]のいずれかに記載の方法により製造される網膜色素上皮細胞。
[28]上記[1]~[22]、[21’]及び[22’]のいずれかに記載の方法により製造される網膜色素上皮細胞を有効成分として含有する、医薬組成物。
本発明において「多能性幹細胞」とは、自己複製能と多能性を有する細胞であり、インビトロにおいて培養することが可能で、かつ、三胚葉(外胚葉、中胚葉、内胚葉)に属する細胞系列すべてに分化しうる能力(多能性(pluripotency))を有する幹細胞をいう。
人工多能性幹細胞は、2006年、山中らによりマウス細胞で初めて樹立され(Cell, 2006, 126(4) pp.663-676)、2007年にはヒト線維芽細胞でも樹立された(Cell, 2007, 131(5) pp.861-872;Science, 2007, 318(5858) pp.1917-1920;Nat. Biotechnol., 2008, 26(1) pp.101-106)。人工多能性幹細胞の誘導方法についてはその後も様々な改良が行われており、具体的な製造方法は、例えば、マウス人工多能性幹細胞については、Cell. 2006 Aug 25;126(4):663-76、ヒト人工多能性幹細胞については、Cell. 2007 Nov 30;131(5):861-72等に記載されている。
人工多能性幹細胞として、遺伝子発現による直接初期化で製造する方法以外に、化合物の添加などにより体細胞より人工多能性幹細胞を誘導することもできる(Science, 2013, 341 pp. 651-654)。
また、株化された人工多能性幹細胞を入手する事も可能であり、例えば、京都大学で樹立された201B7細胞、201B7-Ff細胞、253G1細胞、253G4細胞、1201C1細胞、1205D1細胞、1210B2細胞又は1231A3細胞等のヒト人工多能性細胞株が、京都大学又はiPSアカデミアジャパン株式会社より入手可能である。また、株化された人工多能性幹細胞として、例えば、京都大学で樹立されたFf-I01細胞、Ff-I14細胞及びQHJI01細胞が、京都大学より入手可能である。
選別した細胞株の中から目的とする相同組換え体を選択する方法としては、ゲノムDNAに対するサザンハイブリダイゼーション法やPCR法等があげられる。
「接着培養」とは、細胞又は細胞の凝集体を培養器材等に接着させる条件で行われる培養をいう。この場合、細胞が接着するとは、細胞または細胞の凝集体と培養器材の間に、強固な細胞-基質間結合ができることをいう。すなわち、接着培養とは、細胞または細胞の凝集体と培養器材等との間に強固な細胞-基質間結合を作らせる条件での培養をいう。
浮遊培養中の細胞の凝集体では、細胞と細胞が面接着(plane attachment)する。浮遊培養中の細胞の凝集体では、細胞-基質間結合が培養器材等との間にはほとんど形成されないか、あるいは、形成されていてもその寄与が小さい。一部の態様では、浮遊培養中の細胞の凝集体では、内在の細胞-基質間結合が凝集塊の内部に存在するが、細胞-基質間結合が培養器材等との間にはほとんど形成されないか、あるいは、形成されていてもその寄与が小さい。細胞と細胞が面接着するとは、細胞と細胞が面で接着することをいう。より詳細には、細胞と細胞が面接着するとは、ある細胞の表面積のうち別の細胞の表面と接着している割合が、例えば、1%以上、好ましくは3%以上、より好ましくは5%以上であることをいう。細胞の表面は、膜を染色する試薬(例えばDiI)による染色や、細胞接着因子(例えば、E-cadherinやN-cadherin)の免疫染色により、観察できる。
血清の濃度は、網膜色素上皮細胞を効率的に分化誘導しうる濃度であれば特に限定されないが、例えば約0.5%~30%(v/v)の範囲から適宜設定できる。また、当該濃度は一定でもよく、段階的に変化させてもよい。
「無血清条件」とは、無調整又は未精製の血清を含まない条件、具体的には、無血清培地を使用する条件を意味する。
例えば、「FGFシグナル伝達経路作用物質を含む培地」とは、外来性のFGFシグナル伝達経路作用物質が添加された培地または外来性のFGFシグナル伝達経路作用物質を含む培地である。
本発明の製造方法は、下記工程(1)~(2)を含む、網膜色素上皮細胞の製造方法である:
(1)多能性幹細胞を、FGF受容体阻害物質及び/又はMEK阻害物質を含む培地で、30日を超えない期間培養する第一工程、及び
(2)第一工程で得られた細胞を、Nodalシグナル伝達経路阻害物質及び/又はWntシグナル伝達経路阻害物質の存在下において培養し、網膜色素上皮細胞を形成させる第二工程。
第一工程における好ましい多能性幹細胞として、人工多能性幹細胞、更に好ましくはヒト人工多能性幹細胞が挙げられる。ここで人工多能性幹細胞の製造方法には特に限定はなく、上述のとおり当業者に周知の方法で製造することができるが、人工多能性幹細胞の作成工程(すなわち、体細胞を初期化し多能性幹細胞を樹立する工程)もフィーダーフリーで行うことが望ましい。
当業者に汎用されている未分化維持因子としては、プライムド多能性幹細胞(Primed pluripotent stem cells)(例えば、ヒトES細胞やヒトiPS細胞)の場合、FGFシグナル伝達経路作用物質、TGFβファミリーシグナル伝達経路作用物質等を挙げることができる。FGFシグナル伝達経路作用物質として具体的には、線維芽細胞増殖因子(例えば、bFGF、FGF4やFGF8)が挙げられる。また、TGFβファミリーシグナル伝達経路作用物質としては、TGFβシグナル伝達経路作用物質(例えばTGFβ1、TGFβ2)、Nodal/Activinシグナル伝達経路作用物質(例えばNodal、Activin A、Activin B)が挙げられる。ヒト多能性幹細胞(ヒトES細胞、ヒトiPS細胞)を培養する場合、未分化維持因子は好ましくはbFGFである。
細胞保護剤添加処理に用いられる細胞保護剤としては、ヘパリン、血清、又は血清代替物を挙げることができる。また、分散により誘導される細胞死(特に、ヒト多能性幹細胞の細胞死)を抑制するために、分散の際に、ROCK阻害物質を添加してもよい。ROCK阻害物質としては、Y-27632、Fasudil(HA1077)、H-1152等を挙げることができる。
細胞分散液処理に用いられる細胞分散液としては、トリプシン、コラゲナーゼ、ヒアルロニダーゼ、エラスターゼ、プロナーゼ、DNase又はパパイン等の酵素類や、エチレンジアミン四酢酸等のキレート剤のいずれかを含む溶液を挙げることができる。市販の細胞分散液、例えば、TrypLE Select (Life Technologies社製)やTrypLE Express (Life Technologies社製)を用いることもできる。
機械的分散処理の方法としては、ピペッティング処理又はスクレーパーでの掻き取り操作が挙げられる。
分散された多能性幹細胞を、新たな培養容器に播種し、第一工程に供することができる。
分散により誘導される細胞死(特に、ヒト多能性幹細胞の細胞死)を抑制するために、多能性幹細胞を、新たな培養容器に播種した後に、ROCK阻害物質存在下で維持培養を継続し、その後第一工程を開始してもよい。ROCK阻害物質処理の期間は、分散により誘導される細胞死が抑制できる限り特に限定されないが、通常、12~24時間程度である。
本発明の方法により網膜色素上皮細胞を製造可能な範囲であれば、当該濃度は第一工程を通して一定でもよく、段階的に変化させてもよい。
培地交換操作に用いる道具は特に限定されないが、例えば、ピペッター、マイクロピペット、マルチチャンネルマイクロピペット、連続分注器、などが挙げられる。例えば、培養容器として96ウェルプレートを用いる場合、マルチチャンネルピペットマンを使ってもよい。
所定の培養条件において所与の培養期間が「少なくとも1つの眼形成初期のマーカー、具体的には眼形成転写因子の発現を誘導するのに十分な期間」または「PAX6、LHX2及びSIX3の少なくとも1つの遺伝子発現を誘導するのに十分な期間」であるか否かは、当該条件における当該期間の培養後の細胞集団において、無処理対照と比べてこれらの少なくとも1つの遺伝子発現が有意に検出されるかどうかを確認することにより決定することができる。当業者であれば、例えば、ノザンブロット、RT-PCR、マイクロアレイ等の手法により、これらの遺伝子の発現を検出する事ができる。
(1)眼形成転写因子の少なくとも1つの遺伝子発現を誘導するのに十分な期間、多能性幹細胞を、FGF受容体阻害物質及び/又はMEK阻害物質を含む培地で培養する第一工程、及び
(2)第一工程で得られた細胞を、Nodalシグナル伝達経路阻害物質及び/又はWntシグナル伝達経路阻害物質の存在下において培養し、網膜色素上皮細胞を形成させる第二工程。
(1)PAX6、LHX2及びSIX3の少なくとも1つの遺伝子発現を誘導するのに十分な期間、多能性幹細胞を、FGF受容体阻害物質及び/又はMEK阻害物質を含む培地で培養する第一工程、及び
(2)第一工程で得られた細胞を、Nodalシグナル伝達経路阻害物質及び/又はWntシグナル伝達経路阻害物質の存在下において培養し、網膜色素上皮細胞を形成させる第二工程。
第一工程で得られた細胞を、Nodalシグナル伝達経路阻害物質及び/又はWntシグナル伝達経路阻害物質の存在下において培養し、網膜色素上皮細胞を形成させる第二工程について説明する。
本発明の方法により網膜色素上皮細胞を製造可能な範囲であれば、当該濃度は第二工程を通して一定でもよく、段階的に変化させてもよい。
培地交換操作に用いる道具は特に限定されないが、例えば、ピペッター、マイクロピペット、マルチチャンネルマイクロピペット、連続分注器、などが挙げられる。例えば、培養容器として96ウェルプレートを用いる場合、マルチチャンネルピペットを使ってもよい。
また、当業者であれば、細胞マーカー(RPE65(網膜色素上皮細胞)、Mitf(網膜色素上皮細胞)BEST1(網膜色素上皮細胞)、CRALBP(網膜色素上皮細胞)など)の発現や、メラニン顆粒の存在(黒褐色)、細胞間のタイトジャンクション、多角形・敷石状の特徴的な細胞形態などにより、網膜色素上皮細胞の生成を確認する事が可能であり、これらを確認する事で培養期間を設定する事も可能である。
基礎培地としては、上記定義の項で記載したようなものである限り特に限定されない。血清は、ウシ、ヒトなどの哺乳動物に由来する血清を使用できる。本発明においては、目的の細胞の品質管理上の観点から血清代替物を使用する事が好ましく、特に神経細胞培養用血清代替物であるB27が好適である。その他の成分としては、例えば、L-glutamine、ペニシリンナトリウム、硫酸ストレプトマイシン等が挙げられる。
第二工程において、網膜色素上皮細胞を接着培養により製造した場合、網膜色素上皮細胞は互いに接着しシート状構造を採り得る。従って、患者に移植可能な網膜色素上皮細胞のシートを製造する事が可能である。この網膜色素上皮細胞のシートは、網膜疾患を治療する細胞移植治療薬として用いる細胞集団として特に有用である。また、接着培養により製造された網膜色素上皮細胞を、上記方法で剥離させ、網膜色素上皮細胞を独立した単独の細胞として回収し、これらを生理的な水性溶媒(生理食塩水、緩衝液、無血清培地等)に懸濁させることにより、懸濁液として調製する事も可能である。
本発明の製造方法により製造された網膜色素上皮細胞は、健常および疾患のモデル細胞として、網膜系疾患治療薬および糖尿病など他の合併症の疾患治療薬、またはその予防薬のスクリーニング・薬効評価、化学物質等の安全性試験、ストレス試験、毒性試験、副作用試験、感染・混入試験に活用が可能である。一方、網膜細胞特有の光毒性、網膜興奮毒性等の毒性研究、毒性試験等に活用することも可能である。その評価方法としては、アポトーシス評価などの刺激・毒性試験のほか、前駆細胞から網膜色素上皮細胞および視細胞への正常分化に及ぼす影響を評価する試験(各種遺伝子マーカーのRT-PCR、サイトカインのELISAなどによる発現タンパク質解析、貪食能試験)、光毒性などの毒性試験、視機能に対する網膜電位や経上皮電気抵抗、自己免疫反応に起因する細胞傷害試験などがある。また、これらの試験の為の細胞材料としては、網膜色素上皮細胞のみならず、その前駆細胞も用いることが可能で、例えば、細胞を播種接着したプレート、細胞懸濁液、そのシートまたは成形体を提供することができる。これは、ヒトおよび動物試験の外挿試験として用いることができる。
本発明は、本発明の製造方法により製造される網膜色素上皮細胞の有効量を含む医薬組成物を提供する。
本発明の医薬組成物は、本発明の製造方法により製造される網膜色素上皮細胞を、適切な生理的な水性溶媒で懸濁することにより、懸濁液として製造することができる。必要であれば、凍結保存剤を添加して、液体窒素等により凍結保存し、使用時に解凍し、緩衝液で洗浄し、移植医療に用いても良い。
本発明の製造方法で得られる網膜色素上皮細胞を、ピンセット等を用いて適切な大きさに細切し、シート剤とすることもできる。
また、本発明の製造方法で得られる細胞は、分化誘導を行う第二工程で接着培養を行うことにより、シート状の細胞に成形し、シート剤とすることもできる。
本発明の製造方法により製造された網膜色素上皮細胞(上記濃縮及び増幅操作等を経た網膜色素上皮細胞を含む。)は、懸濁液やシート形状により生体へ移植して網膜疾患を治療する細胞移植治療薬として用いることができる。また、本発明は当該治療薬を患者に投与することを含む治療方法も提供する。ここで網膜疾患とは、網膜に関わる眼科疾患であって、糖尿病など他の疾患による合併症も含まれる。本発明における網膜疾患としては、網膜色素上皮細胞の障害に基づく疾患が挙げられ、例えば、加齢性黄斑変性症、網膜色素変性症、糖尿病性網膜症又は網膜剥離等が挙げられる。すなわち、患者における網膜色素上皮細胞の損傷部位に、本発明の製造方法により製造された網膜色素上皮細胞を補充することができる。
即ち、好ましい態様において、本発明の方法において、多能性幹細胞として、レシピエントと免疫が適合する他者の体細胞から樹立した多能性幹細胞から、アロの網膜色素上皮細胞又はこれを含む組織を製造し、これが当該レシピエントに移植される。または、レシピエントの体細胞から樹立した多能性幹細胞(例、誘導多能性幹細胞)を用いることにより、当該レシピエントについて免疫学的自己の網膜色素上皮細胞又はこれを含む組織を製造し、これが当該レシピエントに移植される。
ヒトiPS細胞(201B7株及び1231A3株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03培地(味の素)(以下、AK03培地)、又は、Essential 8培地(Life Technologies)を使用した。
MEK阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり1.2 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]含有AK03培地、又は、Essential 8培地で、37℃、5% CO2条件下で培養した。播種翌日、MEK阻害物質としてPD0325901(SIGMA)を、AK03培地に最終濃度1 μM、又は、Essential 8培地に最終濃度0.03 μMになるよう添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり、AK03培地使用時には2.0 x 105細胞、Essential 8培地使用時には5.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目は、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加したAK03培地、又は、Essential 8培地を使用し、培養2日目から5日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養6日目から9日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養10日目から13日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養14日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。
培養38日目から42日目に培養プレートを観察した結果、AK03培地、及び、Essential 8培地の両培地使用時、201B7株、1231A3株の両株において、黒褐色を呈する細胞集団の広範囲に及ぶ出現を確認できた(図1)。顕微鏡観察により、それら細胞は、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示していた(図2)。
ヒトiPS細胞(201B7株及び1231A3株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03培地(味の素)(以下、AK03培地)、又は、Essential 8培地(Life Technologies)を使用した。
MEK阻害物質処理工程を含まない網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE selectで処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり、AK03培地使用時には2.0 x 105細胞、Essential 8培地使用時には5.0 x 105細胞播種し、37℃、5% CO2条件下で培養した。培養1日目は、ROCK阻害物質としてY-27632(和光純薬)(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加したAK03培地、又は、Essential 8培地を使用し、培養2日目から5日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養6日目から9日目は、15% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養10日目から13日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養14日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。
培養38日目から42日目に培養プレートを観察した結果、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現は、わずかしか確認できなかった(図3)。
ヒトiPS細胞(201B7株及び1231A3株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03培地(味の素)(以下、AK03培地)、又は、Essential 8培地(Life Technologies)を使用した。
FGF受容体阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり1.2 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]含有AK03培地、又は、Essential 8培地で、37℃、5% CO2条件下で培養した。播種翌日、FGF受容体阻害物質としてPD173074(SIGMA)を最終濃度100 nMになるようAK03培地、又は、Essential 8培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。一例においては、未分化維持因子(bFGF)を含まない培地におけるFGF受容体阻害物質の効果を検討するために、bFGFを添加しないStemSure hPSC Medium Δ(和光純薬)(以降、StemSure hPSC Medium Δ w/o bFGF)を使用した。すなわち、StemSure hPSC Medium Δ w/o bFGFに、FGF受容体阻害物質としてPD173074(SIGMA)を最終濃度100 nMになるように添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり、AK03培地、又は、StemSure hPSC Medium Δ w/o bFGF使用時には2.0 x 105細胞、Essential 8培地使用時には5.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目は、ROCK阻害物質としてY-27632(和光純薬)(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加したAK03培地、Essential8培地、又は、StemSure hPSC Medium Δ w/o bFGFを使用し、培養2日目から5日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養6日目から9日目は、15% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養10日目から13日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養14日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。
培養38日目から42日目に培養プレートを観察した結果、AK03培地、Essential 8培地、又は、StemSure hPSC Medium Δ w/o bFGF使用時において、201B7株、及び/又は、1231A3株において、黒褐色を呈する細胞集団の広範囲に及ぶ出現を確認できた(図4)。顕微鏡観察により、それら細胞は、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示していた(図5)。
ヒトiPS細胞(201B7株及び1231A3株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。「StemFit(登録商標)」AK03培地(味の素)(以下、AK03培地)、又は、Essential 8培地(Life Technologies)を使用した。
FGF受容体阻害物質処理工程を含まない網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞、又は、StemSure hPSC Medium Δ w/o bFGF(和光純薬)で6日間培養したiPS細胞を0.5 x TrypLE selectで処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり、AK03培地、又は、StemSure hPSC Medium Δ w/o bFGF使用時には2.0 x 105細胞、Essential 8培地使用時には5.0 x 105細胞播種し、37℃、5% CO2条件下で培養した。培養1日目は、ROCK阻害物質としてY-27632(和光純薬)(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加したAK03培地、Essential 8培地、又は、StemSure hPSC Medium Δw/o bFGFを使用し、培養2日目から5日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養6日目から9日目は、15% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養10日目から13日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養14日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。
培養38日目から42日目に培養プレートを観察した結果、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現は、わずかしか確認できなかった(図6)。
ヒトiPS細胞(201B7株及び1231A3株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03培地(味の素)(以下、AK03培地)を使用した。
MEK阻害物質及び/又はFGF受容体阻害物質と、各種阻害物質、シグナル伝達経路阻害物質又はシグナル伝達経路作用物質との組み合わせ処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE selectで処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり1.2 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]含有AK03培地で、37℃、5% CO2条件下で培養した。細胞播種翌日、MEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)、FGF受容体阻害物質としてPD173074(SIGMA)(最終濃度100 nM)、BMP受容体阻害物質としてLDN193189(STEMGENT)(最終濃度100 nM)、Shhシグナル伝達経路作用物質としてSAG(Enzo Life Sciences)(最終濃度30 nM)、PKC阻害物質としてGo6983(SIGMA)(最終濃度2 μM)を図7に示す組み合わせで培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目は、ROCK阻害物質としてY-27632(和光純薬)(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加したAK03培地を使用し、培養2日目から5日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養6日目から9日目は、15% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養10日目から13日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養14日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。同時に、MEK阻害物質処理工程を含む実施例1、MEK阻害物質処理工程を含まない比較例1、FGF受容体阻害物質処理工程を含む実施例2、および、FGF受容体阻害物質処理工程を含まない比較例2の条件でも実験を実施した。
培養38日目から47日目に培養プレートを観察した結果、比較例1及び比較例2の条件では、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現がわずかであった(図7、無処理)。それに対し、実施例1のMEK阻害物質処理工程を含む製造条件、実施例2のFGF受容体阻害物質処理工程を含む製造条件、及び、MEK阻害物質及び/又はFGF受容体阻害物質と、各種阻害物質、シグナル伝達経路阻害物質又はシグナル伝達経路作用物質との組み合わせ処理工程を含む製造条件では、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示すRPE細胞の広範囲な出現を確認できた(図7)。ウェル全体に占めるRPE細胞の割合を目視で判定し、割合に応じて0から5の6段階に分けた場合(図8A)、図8B及び8Cに示す全ての化合物組み合わせ処理条件において、無処理よりも高い割合のRPE細胞の出現を確認できた(図8B及び8C)。
ヒトiPS細胞(Ff-I01株及びQHJI01株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03N培地(味の素)(以下、AK03N培地)を使用した。
MEK阻害物質又はFGF受容体阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]含有AK03N培地で、37℃、5% CO2条件下で培養した。播種翌日、MEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)、又は、FGF受容体阻害物質としてPD173074(SIGMA)(最終濃度100 nM)をAK03N培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目から4日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養5日目から8日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養9日目から12日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養13日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。同時に、MEK阻害物質処理工程を含まない比較例1(ただし、AK03培地はAK03N培地に変更)、及び、FGF受容体阻害物質処理工程を含まない比較例2の条件(ただし、AK03培地はAK03N培地に変更)でも実験を実施した。
培養43日目に培養プレートを観察した結果、比較例1及び比較例2の条件では、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現はほぼ見られなかった(図9、無処理)。一方、MEK阻害物質及びFGF受容体阻害物質で暴露した場合、Ff-I01株、QHJI01株の両株において、黒褐色を呈する細胞集団の広範囲な出現を確認できた(図9、MEKi、FGFRi)。
ヒトiPS細胞(QHJI01株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03N培地(味の素)(以下、AK03N培地)を使用した。
MEK阻害物質又はFGF受容体阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)] 含有AK03N培地で、37℃、5% CO2条件下で培養した。播種翌日、2日後、3日後、4日後、5日後、又は、6日後に、MEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)、又は、FGF受容体阻害物質としてPD173074(SIGMA)(最終濃度100 nM)をAK03N培地に添加し(第一工程開始)、6日間、5日間、4日間、3日間、2日間、又は、1日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目から4日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養5日目から8日目は、15% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養9日目から12日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養13日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。
培養48日目に培養プレートを観察した結果、MEK阻害物質及びFGF受容体阻害物質の両処理において、暴露日数2日間以上で黒褐色を呈する細胞集団の増加が見られ、暴露日数6日間までの間、暴露日数の増加に伴いウェル全体に占める呈色細胞の割合が増加した(図10)。特に暴露日数4日間~6日間で黒褐色を呈する細胞集団の顕著な増加が見られた。
ヒトiPS細胞(1231A3株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03培地(味の素)(以下、AK03培地)を使用した。
MEK阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり1.2 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]を含むAK03培地で、37℃、5% CO2条件下で培養した。播種翌日、4日後、又は、6日後に、MEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)をAK03培地に添加し(第一工程開始)、6日間、3日間、又は、1日間暴露した(第一工程終了)。また、MEK阻害物質6日間暴露した細胞を0.5 x TrypLE selectで処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり0.8 x 104細胞播種し、Y-27632(最終濃度10 μM)、及び、PD0325901(最終濃度1 μM)含有AK03培地で、37℃、5% CO2条件下で更に7日間培養することで、13日間 MEK阻害物質で暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり1.2 x 106細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目は、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加したAK03培地を使用し、培養2日目から5日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養6日目から9日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養10日目から13日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養14日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。同時に、MEK阻害物質処理工程を含まない比較例1の条件でもRPE細胞の製造を実施した。
培養37日目に培養プレートを観察した結果、比較例1の条件、及び、MEK阻害物質暴露1日間では、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現はほぼ見られなかった(図11、無処理、MEKi 1日間)。一方、MEK阻害物質暴露3日間、6日間では、黒褐色を呈する細胞集団の広範囲な出現を確認でき(図11、MEKi 3日間、6日間)、暴露日数13日間においても同様に、十分な呈色細胞集団の出現が確認された(図11、MEKi 13日間)。
ヒトiPS細胞(Ff-I01株及びQHJI01株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03N培地(味の素)(以下、AK03N培地)を使用した。
MEK阻害物質又はFGF受容体阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]含有AK03N培地で、37℃、5% CO2条件下で培養した。播種翌日、又は、4日後にMEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)、又は、FGF受容体阻害物質としてPD173074(SIGMA)(最終濃度100 nM)をAK03N培地に添加し(第一工程開始)、6日間、又は、3日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、第二工程に移す細胞分以外はマイクロアレイ用検体としてRNA抽出に使用し、第二工程移行分の細胞は、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目は、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加したAK03培地、又は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542(和光純薬)(最終濃度5 μM)、CKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]を使用した。培養1日目にAK03培地(Y-27632、SB-431542、及び、CKI-7含有)を使用した場合、培養2日目から5日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542(和光純薬)(最終濃度5 μM)、CKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養6日目から9日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養10日目から13日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養14日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培養1日目に基礎培地(20%KSR、Y-27632、SB-431542、及び、CKI-7含有)を使用した場合、培養2日目から4日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養5日目から8日目は、15% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養9日目から12日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養13日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地を使用した。培地は毎日、全量交換した。同時に、MEK阻害物質処理工程を含まない比較例1の条件(AK03培地はAK03N培地に変更)でもマイクロアレイ用検体の回収とRPE細胞の製造を実施した。
RNA抽出にはRNeasy Mini Kit(QIAGEN)を使用し、GeneChip Human Genome U133 Plus 2.0 Array(Affymetrix)を用いてマイクロアレイ解析を実施した。マイクロアレイ解析は倉敷紡績株式会社の受託解析を利用した。
培養43日目の培養プレートの観察像を元に、図8Aに従い、ウェル全体に占めるRPE細胞の割合を目視判定した結果(RPE細胞の割合に応じて0から5の6段階にスコア化)と、マイクロアレイ解析の結果である第一工程終了時における眼形成初期マーカーPAX6、LHX2、SIX3の発現値(Signal)、及び、フラグ(Detection)を表にまとめた(図12)。フラグは発現値の信頼性を表しており、Pは信頼性が高く、Aは信頼性が低いことを意味する。この結果から、ウェル全体に占めるRPE細胞の割合と、第一工程終了時におけるPAX6、LHX2、SIX3の発現値の間に相関関係が認められた。従って、第二工程への移行時期を、これら遺伝子の発現に基づき決定できる事が判明した。
ヒトiPS細胞(QHJI01株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03N培地(味の素)(以下、AK03N培地)を使用した。
MEK阻害物質又はFGF受容体阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]含有AK03N培地で、37℃、5% CO2条件下で培養した。播種翌日、MEK阻害物質としてPD0325901(SIGMA)を最終濃度0.25 μM、0.5 μM、1 μM、2 μM、又は、4 μM、FGF受容体阻害物質としてPD173074(SIGMA)を最終濃度25 nM、50 nM、100 nM、200 nM、又は、400 nMになるようAK03N培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目から4日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養5日目から8日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養9日目から12日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養13日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。同時に、MEK阻害物質処理工程を含まない比較例1(AK03培地はAK03N培地に変更)、及び、FGF受容体阻害物質処理工程を含まない比較例2の条件(AK03培地はAK03N培地に変更)でもRPE細胞の製造を実施した。
培養36日目と49日目に培養プレートを観察した結果、比較例1及び比較例2の条件では、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現はほぼ見られなかった(図13、無処理)。一方、検討した全てのMEK阻害物質濃度(0.25~4 μM)、及び、FGF受容体阻害物質濃度(25~400 nM)において、黒褐色を呈する細胞集団の広範囲な出現を確認できた(図13、MEKi:0.25~4 μM、FGFRi:25~400 nM)。
ヒトiPS細胞(QHJI01株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03N培地(味の素)(以下、AK03N培地)を使用した。
MEK阻害物質又はFGF受容体阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]を含むAK03N培地で、37℃、5% CO2条件下で培養した。播種翌日、MEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)、又は、FGF受容体阻害物質としてPD173074(SIGMA)(最終濃度100 nM)をAK03N培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり0.2、0.4、0.6、1.0、2.0、又は、4.0 x 105細胞(0.2、0.4、0.6、1.0、2.0、又は、4.0 x 104細胞/cm2)播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目から4日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養5日目から8日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養9日目から12日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養13日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。
培養49日目に培養プレートを観察した結果、検討した全ての播種細胞数(0.2、0.4、0.6、1.0、2.0、又は、4.0 x 104細胞/cm2)において、黒褐色を呈する細胞集団の広範囲な出現を確認できた(図14)。
ヒトiPS細胞(QHJI01株及び1231A3株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03N培地(味の素)(以下、AK03N培地)を使用した。
MEK阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]を含むAK03N培地で、37℃、5% CO2条件下で培養した。播種翌日、MEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)、PD184352(SIGMA)(最終濃度1.5 μM、3 μM、6 μM)、U0126(SIGMA)(最終濃度5 μM、10 μM)、TAK-7331(Selleck)(最終濃度0.3 μM)、又は、AZD-8330(Selleck)(最終濃度0.3 μM)をAK03N培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目から4日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養5日目から8日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養9日目から12日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養13日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。同時に、MEK阻害物質処理工程を含まない比較例1の条件(AK03培地はAK03N培地に変更)でもRPE細胞の製造を実施した。
培養49日目と50日目に培養プレートを観察した結果、比較例1の条件では、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現はほぼ見られなかった(図15、無処理)。一方、検討した全てのMEK阻害物質(PD0325901、PD184352、U0126、TAK-7331、AZD-8330)において、黒褐色を呈する細胞集団の広範囲な出現を確認できた(図15、PD0325901、PD184352、U0126、TAK-733、AZD-8330)。
ヒトiPS細胞(QHJI01株及び1231A3株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03N培地(味の素)(以下、AK03N培地)を使用した。
FGF受容体阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]を含むAK03N培地で、37℃、5% CO2条件下で培養した。播種翌日、FGF受容体阻害物質としてPD173074(SIGMA)(最終濃度100 nM)、又は、SU5402(SIGMA)(最終濃度5 μM、10 μM、20 μM)をAK03N培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目から4日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養5日目から8日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養9日目から12日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養13日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。同時に、FGF受容体阻害物質処理工程を含まない比較例2の条件(AK03培地はAK03N培地に変更)でもRPE細胞の製造を実施した。
培養49日目に培養プレートを観察した結果、比較例2の条件では、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現はほぼ見られなかった(図16、無処理)。一方、検討した全てのFGF受容体阻害物質(PD173074、SU5402)において、黒褐色を呈する細胞集団の広範囲な出現を確認できた(図16、PD173074、SU5402)。
ヒトiPS細胞(QHJI01株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03N培地(味の素)(以下、AK03N培地)を使用した。
MEK阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]を含むAK03N培地で、37℃、5% CO2条件下で培養した。播種翌日、MEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)をAK03N培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした12穴培養プレートに、1穴あたり0.8 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。第二工程では以下の3種類の培地を使用した。培養1日目から12日目に、10% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)](図17、NODALi+WNTi)、10% KSR、Y-27632(最終濃度10 μM) 、SB-431542 (最終濃度5 μM) を添加した基礎培地(図17、NODALi)、又は、10% KSR、Y-27632(最終濃度10 μM) 、CKI-7 (最終濃度3 μM)を添加した基礎培地(図17、WNTi)を使用した。培養13日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。
培養43日目に培養プレートを観察した結果、Nodalシグナル伝達経路阻害物質とWntシグナル伝達経路阻害物質の両処理条件(NODALi+WNTi)、及び、Nodalシグナル伝達経路阻害物質の単剤処理条件(NODALi)で同程度の黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現が確認された(図17、NODALi+WNTi、NODALi)。また、Wntシグナル伝達経路阻害物質の単剤処理(WNTi)では、ウェル全体に占める黒褐色面積の減少は見られたが、十分な数のRPE細胞の特徴を示す細胞の出現を確認できた(図17、WNTi)。以上の結果から、第二工程では、Nodalシグナル伝達経路阻害物質、又は、Wntシグナル伝達経路阻害物質のどちらか一方が存在していればよいことが確認できた。
ヒトiPS細胞(201B7株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03培地(味の素)(以下、AK03培地)を使用した。
MEK阻害物質及びBMP受容体阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり1.2 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]含有AK03培地で、37℃、5% CO2条件下で培養した。MEK阻害物質を6日間、BMP受容体阻害物質を1日間暴露する場合は、播種翌日、MEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)をAK03培地に添加し(第一工程開始)、播種6日後にBMP受容体阻害物質としてLDN193189(STEMGENT)(最終濃度100 nM)をAK03培地に添加することで、MEK阻害物質、BMP受容体阻害物質をそれぞれ6日間、1日間暴露した(第一工程終了)。MEK阻害物質、BMP受容体阻害物質を共に6日間暴露する場合は、播種翌日、PD0325901(最終濃度1 μM)とLDN193189(最終濃度100 nM)をAK03培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目は、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加したAK03培地を使用し、培養2日目から5日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、SB-431542 (最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養6日目から9日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養10日目から13日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養14日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。同時に、MEK阻害物質処理工程を含まない比較例1の条件でもRPE細胞の製造を実施した。
培養39日目に観察後、細胞を回収してRNA抽出を行い、リアルタイムRT-PCRを実施した。RNA抽出にはRNeasy Mini Kit(QIAGEN)を使用し、リアルタイムRT-PCRにはQuantiTect Probe RT-PCR Kit(QIAGEN)を使用した。BEST1(Hs00188249_m1)、MITF(Hs01117294_m1)、RAX(Hs00429459_m1)、GAPDH(Hs02758991_g1)のプライマー、プローブはApplied Biosystemsから購入した。各検体のBEST1、MITF、RAXの発現量をGAPDHの発現量で補正し、未分化維持培養条件で培養されていたiPS細胞(未分化)の発現量を1とした時の相対値で表した。
培養39日目の培養プレートの観察像を元に、図8Aに従い、ウェル全体に占めるRPE細胞の割合を目視判定した。その結果、無処理は「1」、MEK阻害物質6日間+BMP受容体阻害物質1日間暴露は「3」、MEK阻害物質6日間+BMP受容体阻害物質6日間暴露は「5」であった(図18上側、細胞写真)。これら検体と未分化維持培養を継続していたiPS細胞(未分化)との間で、網膜色素上皮細胞マーカーであるBEST1、MITF、眼形成初期マーカーであるRAXの発現量をリアルタイムRT-PCR法によって比較した結果、黒褐色細胞の面積と上記マーカー遺伝子の発現量との間に相関関係が認められた(図18下側、グラフ)。以上の結果から、黒褐色を呈している細胞が網膜色素上皮細胞マーカー遺伝子及び眼形成初期マーカー遺伝子を発現していることが示唆された。併せて、本製造法によってRPE細胞が製造された事が遺伝子発現レベルにおいても検証された。
ヒトiPS細胞(1231A3株)のフィーダーフリー条件下での未分化維持培養は、「Nakagawa, M. et. al., Sci. Rep. 2014 Jan 8; 4: 3594」に記載の方法に従い行った。培地は「StemFit(登録商標)」AK03N培地(味の素)(以下、AK03N培地)を使用した。
MEK阻害物質又はFGF受容体阻害物質処理工程を含む網膜色素上皮(RPE)細胞の製造は以下の通り行った。未分化維持培養していたiPS細胞を0.5 x TrypLE select(TrypLE select(Life Technologies)と0.5 mM EDTA/PBS(-)を等量混合)で処理後、セルスクレーパーを用いて剥離し、ピペッティングで単一分散後、iMatrix-511(ニッピ)(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 104細胞播種し、ROCK阻害物質[10 μM Y-27632(和光純薬)]含有AK03N培地で、37℃、5% CO2条件下で培養した。播種翌日、MEK阻害物質としてPD0325901(SIGMA)(最終濃度1 μM)、又は、FGF受容体阻害物質としてPD173074(SIGMA)(最終濃度100 nM)をAK03N培地に添加し(第一工程開始)、6日間暴露した(第一工程終了)。その後、細胞を0.5 x TrypLE selectで処理し、セルスクレーパーを用いて剥離、ピペッティングで単一分散後、iMatrix-511(0.5 μg/cm2)でコーティングした6穴培養プレートに、1穴あたり2.0 x 105細胞播種し、37℃、5% CO2条件下で培養した(第二工程開始)。培養1日目から4日目は、20% KSR(Life Technologies)、Y-27632(最終濃度10 μM)、Nodalシグナル伝達経路阻害物質としてSB-431542(和光純薬)(最終濃度5 μM)、Wntシグナル伝達経路阻害物質としてCKI-7(SIGMA)(最終濃度3 μM)を添加した基礎培地[GMEM培地(SIGMA)、0.1 mM MEM非必須アミノ酸溶液(Life Technologies)、1 mM ピルビン酸ナトリウム(SIGMA)、0.1 mM 2-メルカプトエタノール(和光純薬)、2 mM L-glutamine(SIGMA)、100 U/ml ペニシリン-100 μg/ml ストレプトマイシン(Life Technologies)]、培養5日目から8日目は、15% KSR、 Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養9日目から12日目は、10% KSR、Y-27632(最終濃度10 μM)、SB-431542(最終濃度5 μM)、CKI-7(最終濃度3 μM)を添加した基礎培地、培養13日目から30日目は、10% KSRのみを添加した基礎培地、培養31日目以降は、RPE維持培地 [67% DMEM low glucose(SIGMA)、29% F12(SIGMA)、1.9% B-27 supplement(Life Technologies)、1.9 mM L-glutamine、96 U/ml ペニシリン-96 μg/mlストレプトマイシン]を使用した。培地は毎日、全量交換した。同時に、MEK阻害物質処理工程を含まない比較例1の条件(AK03培地はAK03N培地に変更)でも実験を実施した。
培養43日目に観察後、細胞を回収してRNA抽出を行い、RT-PCRを実施した。RNA抽出にはRNeasy Micro Kit(QIAGEN)、逆転写反応にはOligo(dT)12-18 Primer(Invitrogen)、SuperScript III Reverse Transcriptase(Invitrogen)、PCRにはBlend Taq -Plus-(TOYOBO)を使用した。RPE65、BEST1、CRLBP、GAPDHのプライマー配列は以下の通りである。RPE65-F: TCCCCAATACAACTGCCACT(配列番号1)、RPE65-R: CCTTGGCATTCAGAATCAGG(配列番号2)、BEST1-F: TAGAACCATCAGCGCCGTC(配列番号3)、BEST1-R: TGAGTGTAGTGTGTATGTTGG(配列番号4)、CRALBP-F: GAGGGTGCAAGAGAAGGACA(配列番号5)、CRALBP-R: TGCAGAAGCCATTGATTTGA(配列番号6)、GAPDH-F: ACCACAGTCCATGCCATCAC(配列番号7)、GAPDH-R: TCCACCACCCTGTTGCTGTA(配列番号8)。PCR反応のサイクル数は、RPE65、BEST1、GAPDHが30サイクル、CRALBPは35サイクルで実施した。PCR産物はアガロースゲル電気泳動によって、RPE65は369 bp付近、BEST1は261 bp付近、CRALBPは341 bp付近、GAPDHは452 bp付近に一本のバンドとして検出された。陽性対照としてprimary human RPE(hRPE)、陰性対照として未分化維持培養条件で培養されていたiPS細胞(未分化iPSC)を使用した。
培養43日目に培養プレートを観察した結果、比較例1の条件では、黒褐色、多角、敷石状形態といったRPE細胞の典型的な特徴を示す細胞の出現は、わずかしか確認できなかった(図19右側、細胞写真、無処理)。一方、MEK阻害物質及びFGF受容体阻害物質で暴露した場合、黒褐色を呈する細胞集団の広範囲な出現を確認できた(図19右側、細胞写真、MEKi、FGFRi)。RT-PCRを実施した結果、比較例1の条件では、網膜色素上皮細胞マーカーであるRPE65、BEST1、CRALBPのバンドが非常に薄かった(図19左側、電気泳動図、無処理)。一方、MEK阻害物質及びFGF受容体阻害物質で暴露した検体では明瞭なバンドを確認できた(図19左側、電気泳動図、MEKi、FGFRi)。以上の結果より、MEK阻害物質及びFGF受容体阻害物質処理工程を含む製法によって、RPE細胞が高効率に製造された事が遺伝子発現レベルにおいても検証された。
Claims (28)
- 以下の工程を含む、網膜色素上皮細胞の製造方法:
(1)多能性幹細胞を、FGF受容体阻害物質及び/又はMEK阻害物質を含む培地で、30日を超えない期間培養する第一工程、及び
(2)第一工程で得られた細胞を、Nodalシグナル伝達経路阻害物質及び/又はWntシグナル伝達経路阻害物質の存在下において培養し、網膜色素上皮細胞を形成させる第二工程。 - 第一工程が、無血清条件下で行われる、請求項1に記載の製造方法。
- 第一工程が、フィーダー細胞非存在下で行われる、請求項1又は2に記載の製造方法。
- 第一工程における培地が、さらに未分化維持因子を含む、請求項1~3のいずれか1項に記載の製造方法。
- 未分化維持因子が、FGFシグナル伝達経路作用物質である、請求項4に記載の製造方法。
- FGFシグナル伝達経路作用物質が、bFGFである、請求項5に記載の製造方法。
- FGF受容体阻害物質が、PD173074及びSU5402からなる群から選択される少なくとも1種である、請求項1~6のいずれか1項に記載の製造方法。
- MEK阻害物質が、PD0325901、PD184352、U0126、TAK-733、及びAZD-8330からなる群から選択される少なくとも1種である、請求項1~7のいずれか1項に記載の製造方法。
- Nodalシグナル伝達経路阻害物質が、ALK4,5,又は7阻害物質である、請求項1~8のいずれか1項に記載の製造方法。
- ALK4,5,又は7阻害物質が、SB431542である、請求項9に記載の製造方法。
- Wntシグナル伝達経路阻害物質が、CKI-7である請求項1~10のいずれか1項に記載の製造方法。
- 多能性幹細胞が、霊長類多能性幹細胞である請求項1~11のいずれか1項に記載の製造方法。
- 多能性幹細胞が、ヒト多能性幹細胞である請求項1~12のいずれか1項に記載の製造方法。
- 第一工程における培地が、さらにBMP受容体阻害物質を含む培地である、請求項1~13のいずれか1項に記載の製造方法。
- BMP受容体阻害物質が、ALK2/3阻害物質である請求項14に記載の製造方法。
- ALK2/3阻害物質が、LDN193189である請求項15に記載の製造方法。
- 第一工程における培地が、さらにソニック・ヘッジホッグシグナル伝達経路作用物質を含む請求項1~16のいずれか1項に記載の製造方法。
- ソニック・ヘッジホッグシグナル伝達経路作用物質が、SAGである請求項17に記載の製造方法。
- 第一工程における培地が、さらにPKC阻害物質を含む請求項1~18のいずれか1項に記載の製造方法。
- PKC阻害物質が、Go6983である請求項19に記載の製造方法。
- 第一工程において、培養期間が2日間~13日間である、請求項1~20のいずれか1項に記載の製造方法。
- 第一工程において、培養期間が4日間~6日間である、請求項1~21のいずれか1項に記載の製造方法。
- 請求項1~22のいずれか1項に記載の方法により製造される網膜色素上皮細胞を含有してなる、被験物質の毒性・薬効評価用試薬。
- 請求項1~22のいずれか1項に記載の方法により製造される網膜色素上皮細胞に被験物質を接触させ、該物質が該細胞に及ぼす影響を検定することを含む、該物質の毒性・薬効評価方法。
- 請求項1~22のいずれか1項に記載の方法により製造される網膜色素上皮細胞を含有してなる、網膜色素上皮細胞の障害に基づく疾患の治療薬。
- 請求項1~22のいずれか1項に記載の方法により製造される網膜色素上皮細胞の有効量を、移植を必要とする対象に移植することを含む、網膜色素上皮細胞の障害に基づく疾患の治療方法。
- 網膜色素上皮細胞の障害に基づく疾患の治療における使用のための、請求項1~22のいずれか1項に記載の方法により製造される網膜色素上皮細胞。
- 請求項1~22のいずれか1項に記載の方法により製造される網膜色素上皮細胞を有効成分として含有する、医薬組成物。
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US15/757,202 US11649432B2 (en) | 2015-09-08 | 2016-09-08 | Method for producing retinal pigment epithelial cells |
ES16844466T ES2922475T3 (es) | 2015-09-08 | 2016-09-08 | Método para producir células epiteliales pigmentarias de la retina |
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IL257794A IL257794B (en) | 2015-09-08 | 2016-09-08 | A method for producing retinal pigment epithelial cells |
JP2017538524A JP6792844B6 (ja) | 2015-09-08 | 2016-09-08 | 網膜色素上皮細胞の製造方法 |
CA2997528A CA2997528C (en) | 2015-09-08 | 2016-09-08 | Method for producing retinal pigment epithelial cells |
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EP22171791.1A EP4092109A1 (en) | 2015-09-08 | 2016-09-08 | Method for producing retinal pigment epithelial cells |
HK18110498.6A HK1251249A1 (zh) | 2015-09-08 | 2018-08-15 | 用於製備視網膜色素上皮細胞的方法 |
US18/295,428 US20230287342A1 (en) | 2015-09-08 | 2023-04-04 | Method for producing retinal pigment epithelial cells |
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WO2020218480A1 (ja) | 2019-04-26 | 2020-10-29 | 国立研究開発法人理化学研究所 | 神経網膜と網膜色素上皮細胞とハイドロゲルとを含む複合体及びその製造方法 |
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WO2022054924A1 (ja) | 2020-09-11 | 2022-03-17 | 大日本住友製薬株式会社 | 移植用組織のための媒体 |
WO2022230977A1 (ja) | 2021-04-30 | 2022-11-03 | 国立研究開発法人理化学研究所 | 網膜色素上皮細胞のひも状凝集体、それを製造するためのデバイスおよび製造方法、ならびに該ひも状凝集体を含有する治療薬 |
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AU2016320991A1 (en) | 2018-04-05 |
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