A method for inducing differentiation of peritoneal mesothelial cells from pluripotent stem cells.

Abstract

The present invention addresses the problem of inducing, from pluripotent stem cells, cells that are functional similar to PMC. Said problem is solved by a method that is for inducing peritoneal mesothelium cells from pluripotent stem cells and that comprises: a first culturing step for culturing pluripotent stem cells in the presence of a basic fibroblast growth factor and a bone morphogenetic factor; a second culturing step for culturing the cells cultured in the first culturing step, in the presence of the bone morphogenetic factor but at a concentration higher than that in the first culturing step, and the basic fibroblast growth factor; and a third culturing step for culturing the cells cultured in the second culturing step in the presence of a vitamin-A compound, a glycogen synthase kinase-3 inhibitor, and an inhibitor of activin kinase-2 and activin kinase-3.

Description

[Technical Field]

[0001] This specification discloses a method for differentiating peritoneal mesothelial cells from pluripotent stem cells, peritoneal mesothelial cells obtained by the said method, and a cell composition containing the said peritoneal mesothelial cells. [Background technology]

[0002] Peritoneal mesothelial cells (PMCs) are mainly elongated, flattened, squamous epithelial-like cells that form a single layer of peritoneal mesothelium within the peritoneal cavity. The peritoneal mesothelial cell layer is known to play a barrier function against microorganisms and tumor cells (Non-Patent Literature 1).

[0003] Furthermore, the peritoneal mesothelium also functions as a permeable barrier, and this property is utilized in peritoneal dialysis. However, prolonged exposure of the peritoneum to dialysate, which is highly osmotic, highly glucose-rich, and acidic, often causes mild chronic inflammation and peritoneal damage, gradually damaging mesothelial cells and potentially leading to fibrosis (Non-Patent Literature 2). As a result, encapsulating peritoneal sclerosis may develop. Although rare, encapsulating peritoneal sclerosis is a fatal complication of peritoneal dialysis, necessitating the discontinuation of peritoneal dialysis and a transition to hemodialysis (Non-Patent Literature 3).

[0004] Mesothelial cells also play a role in maintaining serous homeostasis. Mesothelial cells provide a non-adhesive protective surface that allows organ movement within the serosal cavity (Non-Patent Literature 4). Intraperitoneal adhesions, while some congenital, are generally pathological scars that form between intraperitoneal organs in response to trauma such as surgery (Non-Patent Literature 5). Encapsulating peritoneal sclerosis may also be caused in part by adhesions. Adhesions are lesions that occur because the serosal repair process, which normally re-establishes intact and functional non-adhesive mesothelium within days of injury, breaks down. Therefore, one solution to prevent adhesions is to assist in promoting serosal repair (Non-Patent Literature 6). As a method to assist in peritoneal repair, a method of injecting mesothelial cells into the site of peritoneal injury in non-human animal models has been reported. It has been reported that the injected mesothelial cells attach to and are incorporated into the site when the mesothelium regenerates (Non-Patent Literature 7 to 11).

[0005] The main sources of PMCs reported are the greater omentum (Non-Patent Literature 11), avascular mesentery (Non-Patent Literature 12), and visceral adipose tissue (Non-Patent Literature 13, 14). In addition, bone marrow-derived cells (Non-Patent Literature 15), mesenchymal stem cells (Non-Patent Literature 16, 17), and adipose-derived stem cells (Non-Patent Literature 18) have been reported as cell sources applicable to mesothelial repair.

[0006] The embryological origin of PMC is reported to be mesoderm, similar to the epicardium and pleura (Non-Patent Literature 19). Non-Patent Literature 20 describes the induction of epicardium from induced pluripotent stem cells (iPSCs). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Mutsaers SE. Respirology 2002;7(3):171-191 [Non-Patent Document 2] Krediet RT. Kidney Int 1999;55(1):341-356 [Non-Patent Document 3] Moinuddin Z, Summers A, Van Dellen D, et al. Front Physiol 2014;5:470

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Non-licensed literature 9

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Non-Patent Document 13

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Non-Patent Document 20

Summary of the Invention

Problems to be Solved by the Invention

[0008] However, collecting cells described in Non-Patent Documents 11 to 18 from patients for autotransplantation involves a major invasion. Also, it is difficult to culture and maintain a sufficient number of PMCs to cover a wide area such as the abdominal cavity. Furthermore, in the method described in Non-Patent Document 20, PMCs cannot be induced from iPSCs. An object of the present invention is to induce cells having a function similar to PMCs from pluripotent stem cells.

Means for Solving the Problems

[0009] The present invention may include the following aspects. Item 1. A method for inducing peritoneal mesothelial cells from pluripotent stem cells, comprising: a first culture step of culturing pluripotent stem cells in the presence of basic fibroblast growth factor and a bone morphogenetic factor; a second culture step of culturing the cells cultured in the first culture step in the presence of basic fibroblast growth factor and a bone morphogenetic factor at a concentration higher than that in the first culture step; and a third culture step of culturing the cells cultured in the second culture step in the presence of a vitamin A compound; a glycogen synthase kinase-3 inhibitor; and an inhibitor of activin kinase 2 and activin kinase 3. Item 2. The method according to Item 1, wherein the bone morphogenetic factor is bone morphogenetic factor 4. Item 3. The method according to Item 1 or 2, wherein the glycogen synthase kinase-3 inhibitor is CHIR99021. Item 4. The method according to any one of Items 1 to 3, wherein the inhibitor of activin kinase 2 and activin kinase 3 is LDN193189. Item 5. The method according to any one of Items 1 to 4, wherein the culture period of the first culture step is 36 hours to 60 hours, the culture period of the second culture step is 36 hours to 60 hours, and the culture period of the third culture step is 5 days to 7 days. Item 6. When the induced peritoneal mesothelial cells are transplanted into an animal individual, the pluripotent stem cells are derived from the animal individual or another animal individual immunocompatible with the animal individual. The method according to any one of Items 1 to 5. Item 7. The method according to any one of Items 1 to 6, wherein the pluripotent stem cells are human induced pluripotent stem cells. Section 8. Detaching peritoneal mesothelial cells induced by any of the methods described in Sections 1 to 7 from a plate, and seeding the detached peritoneal mesothelial cells onto a new plate. A method for subculturing peritoneal mesothelial cells, including the method described above. Item 9. Peritoneal mesothelial cells induced by any of the methods described in Items 1 to 7 and passaged by the passage method of Item 8. Item 10. A cell composition comprising peritoneal mesothelial cells as described in Item 9. [Effects of the Invention]

[0010] According to the present invention, it is possible to obtain cells with similar cell permeability and wound healing ability to those of a substance. [Brief explanation of the drawing]

[0011] [Figure 1] A shows the flow of the induction method disclosed herein. B shows the expression of marker genes in iPMCs. Results are expressed as the mean and SD of 6 samples. ** indicates p<0.01 (ANOVA). C shows the results of Western blotting of CK-18 protein and β-actin. [Figure 2] This shows the expression of the CDX2 and TBX1 genes in cells after differentiation induction. Results are expressed as the mean and standard deviation of 6 samples. ** indicates p<0.01 (ANOVA). [Figure 3] A shows the schedule for the iPMC subculturing experiment. B shows phase-contrast microscope images of cells at the end of the third culture step (day 10) and 2 days after subculturing (day 12). C shows the expression of marker genes in iPMCs. Results are expressed as the mean and SD of 6 samples. ** indicates p<0.01 (ANOVA). D shows the results of Western blotting for CK-18 protein and β-actin. [Figure 4] The expression of the CDX2 and TBX1 genes in cells at the end of the third culture step (day 10) and two days after passage (day 12) is shown. Results are expressed as the mean and standard deviation of 6 samples. ** indicates p<0.01 (ANOVA). [Figure 5]A shows the design of the in vitro permeability assay system. B shows the measured concentrations of urea nitrogen, potassium, and albumin in the upper and lower layers of the culture medium. Results are expressed as the mean and SD of 5 samples. C shows the permeability of urea nitrogen, potassium, and albumin. In the graph, "Control" shows data from wells where iPMC was not seeded in the upper layer, and "iPMC" shows data from wells where iPMC was seeded in the upper layer. Results are expressed as the mean and SD of 5 samples. ** indicates p<0.01 (Student's t-test). [Figure 6] A shows phase-contrast microscopy images of the area around the wound site of the scratched cell layer at 0, 24, and 48 hours. B shows the mean and standard deviation of the wound distance around the wound site of the scratched cell layer at 0, 24, and 48 hours for 8 samples. ** indicates p<0.01 (ANOVA). C shows phase-contrast microscopy images of the area around the scratched wound site with and without iPMCs added to the plate containing the scratched cell layer. [Modes for carrying out the invention]

[0012] 1. Method for inducing peritoneal mesothelial cells from pluripotent stem cells The method for inducing peritoneal mesothelial cells from pluripotent stem cells disclosed herein (hereinafter also simply referred to as the "induction method") may include the first culture step, the second culture step, and the third culture step described later.

[0013] Pluripotent stem cells are not limited as long as they maintain pluripotency. Examples of pluripotent stem cells include induced pluripotent stem cells (iPSCs), embryonic stem cells, and germ cell-derived pluripotent stem cells. Preferably, induced pluripotent stem cells are used as pluripotent stem cells. Induced pluripotent stem cells are not limited as long as they are cells that have acquired pluripotency by reprogramming animal somatic cells.

[0014] The somatic cells, embryonic stem cells, and germ cell-derived pluripotent stem cells used to produce induced pluripotent stem cells are not limited as long as they are derived from mammals such as humans, mice, rats, rabbits, cats, dogs, sheep, horses, cattle, goats, and monkeys. Furthermore, when peritoneal mesothelial cells induced by the induction method disclosed herein are used for transplantation into an animal, it is preferable that the somatic cells, embryonic stem cells, or germ cell-derived pluripotent stem cells are derived from the same species of animal as the animal receiving the transplant. It is also preferable that the somatic cells, embryonic stem cells, or germ cell-derived pluripotent stem cells are immunocompatible with the animal receiving the transplant.

[0015] Immunocompatibility can be evaluated by analyzing the types of histocompatibility antigens, such as HLA antigens and MHC antigens. For example, if five or more of the six types of histocompatibility antigens, such as HLA antigens and MHC antigens, match, immunocompatibility can be evaluated.

[0016] Induced pluripotent stem cells can be produced by known methods. For example, if somatic cells are derived from mice, induced pluripotent stem cells can be produced by methods such as those described in Nature Protocols (VOL.4 NO.12 2009) and the improved method for establishing mouse iPS cells (CiRA M&M, published July 17, 2008). Furthermore, if somatic cells are of human origin, induced pluripotent stem cells can be produced by methods such as those described in Current Protocols in Stem Cell Biology June, 2009 (01 June 2009, https: / / doi.org / 10.1002 / 9780470151808.sc04a02s9), Generation of Human induced Pluripotent Stem Cells (CiRA M&M, published March 5, 2009), Human iPS cell culture under feeder-free conditions (CiRA_Ff-iPSC_protocol_Eng_v140310, published March 11, 2014), Method for establishing human iPS cells using episomal vectors (CiRA M&M, published April 4, 2011), and Method for establishing iPS cells from peripheral blood using episomal vectors (CiRA M&M, published January 10, 2013).

[0017] Induced pluripotent stem cells can be obtained from cell banks, etc. For example, the Cell Materials Development Laboratory at the RIKEN BioResource Research Center can provide human-derived induced pluripotent stem cells (HPS0354, HPS0014, HPS0003, HPS0009, HPS0029, HPS0223, etc.), mouse-derived induced pluripotent stem cells (APS0001, APS0002, APS0003, APS0004, APS0005, APS0006, APS0007, etc.), and rabbit-derived induced pluripotent stem cells (APS0008, APS0009, APS00010, APS00011, etc.).

[0018] Embryonic stem cells can also be obtained from cell banks, etc. For example, the Cell Materials Development Laboratory at the RIKEN BioResource Research Center can provide human-derived embryonic stem cells (HES0001, HES0002, HES0003, HES0004, HES0005, HES0006, HES0007, HES0008, HES0017, HES0018, HES0019, HES0020, HES0651, HES0652, HES0653, etc.), mouse-derived embryonic stem cells (AES0015, AES0016, AES0017, AES0018, AES0020, etc.), rabbit-derived embryonic stem cells (AES0174, AES0175, etc.), marmoset-derived embryonic stem cells (AES0166), etc.

[0019] Culture for maintaining pluripotent stem cells can be carried out according to known methods. Induced pluripotent stem cells before differentiation induction are preferably maintained under feeder-free culture conditions. For maintenance culture of induced pluripotent stem cells, for example, if the induced pluripotent stem cells are of human origin, a culture medium such as NutriStem® hPSC XF (BLG, Cat. No. 05-100-1A) can be used as a maintenance medium. In addition, a culture plate such as iMatrix-511 silk (Matrixome, Cat. No. 892021) coated plate can be used.

[0020] Induced pluripotent stem cells can be maintained in a wet incubator maintained at approximately 37°C and a CO2 concentration of approximately 5% using the maintenance medium described above. They can be maintained for approximately 2 to 4 days, preferably 3 days, from the first passage.

[0021] Furthermore, the passage of induced pluripotent stem cells is achieved by detaching the cell aggregate using accutase (M&S TechnoSystems, Cat. No. AT104-100ML) or the like when the induced pluripotent stem cells reach, for example, 70 to 90%, transferring the cell aggregate to a culture plate containing fresh maintenance medium, and performing maintenance culture under the above conditions. The treatment with accutase is, for example, performed at approximately 20 to 28°C for approximately 15 to 40 minutes, preferably 25 to 30 minutes. Also, when seeding pluripotent stem cells, it is preferable to culture them in maintenance medium supplemented with a Rho kinase inhibitor until the pluripotent stem cells engraft on the culture plate. An example of a Rho kinase inhibitor is Y27632 (Focus biomolecules, Cat. No. FCS-10-2301-25). The Rho kinase inhibitor can be added at a concentration that can suppress the differentiation of pluripotent stem cells. For example, it can be added to the maintenance medium to a final concentration of approximately 1 μM to 10 μM, preferably 3 μM to 7 μM, and more preferably 4 μM to 6 μM.

[0022] Depending on the induction method, peritoneal mesothelial cells (hereinafter also referred to as "iPMCs") induced from pluripotent stem cells are not limited as long as they have an expression pattern of biomarker genes equivalent to or similar to that of peritoneal mesothelial cells that spontaneously occur in the body of animals. Examples of biomarker genes for peritoneal mesothelial cells include cytokeratin 18 (CK-18), mesoserine (MSLN), and Wilms' tumor (WT1).

[0023] It is preferable that iPMCs have the same permeability to substances as peritoneal mesothelial cells that naturally occur in the body of animals. Permeability refers to the property that low molecular weight substances such as water, electrolytes (ions) such as sodium, potassium, chloride, phosphorus, and magnesium, urea, uric acid, creatinine, ammonia, amino acids, and glucose can easily pass through, while high molecular weight substances such as proteins such as albumin cannot easily pass through. Preferably, the permeability to low molecular weight substances is, for example, 90% or more, and the permeability to high molecular weight substances is, for example, 50% or less. The permeability to substances can be evaluated by known methods such as permeability tests using Transwell (trademark) (Corning (registered trademark)), etc. Furthermore, it is preferable that iPMCs have a wound repair function. The wound repair function can be evaluated by known methods such as wound healing assays.

[0024] 1-1. First culture step The first culture step includes culturing pluripotent stem cells in the presence of basic fibroblast growth factor and bone morphogenetic factors.

[0025] Before contacting the pluripotent stem cells that have been maintained in culture with basic fibroblast growth factor and osteogenic factors, it is preferable to excavate the cell aggregate using accutase or the like, and pre-culture them in the aforementioned maintenance medium containing a Rho kinase inhibitor until they reach approximately 60 to 80% confluence. An example of a Rho kinase inhibitor is Y27632 (Focus biomolecules, Cat. No. FCS-10-2301-25). The Rho kinase inhibitor can be added at a concentration that can suppress the differentiation of pluripotent stem cells. For example, it can be added to the maintenance medium at a final concentration of approximately 1 μM to 10 μM, preferably approximately 3 μM to 7 μM, and more preferably approximately 4 μM to 6 μM.

[0026] For pre-culturing, plates coated with Matrigel (Corning®, Cat. No. 356237), for example, can be used. Pre-culturing can also be performed in a wet incubator maintained at approximately 37°C and a CO2 concentration of approximately 5%.

[0027] After pre-culture, the first culture step is initiated by replacing the maintenance medium in each plate containing pluripotent stem cells with a medium containing basic fibroblast growth factor and osteogenic factors. This culture step enables the pluripotent stem cells to differentiate into early mesoderm (EM).

[0028] The culture medium for differentiating pluripotent stem cells is not limited as long as it does not hinder the differentiation of pluripotent stem cells into target cells. For example, DMEM Ham's / F12 medium can be used as a basal medium for preparing the culture medium. Since it is preferable not to add serum to the culture medium, it is preferable to add a culture supplement such as B27 (trademark) Serum Free Supplement (Thermo Fisher Scientific) to the basal medium and use it as the culture medium. The amount of culture supplement added is about 0.5 to 4% at the final concentration, preferably about 1 to 3%, and more preferably about 1.5 to 2.5%.

[0029] Basic fibroblast growth factor (bFGF) is also called bFGF or FGF2. Basic fibroblast growth factor is not limited as long as it is a polypeptide that functions as basic fibroblast growth factor. Preferably, it is a recombinant basic fibroblast growth factor polypeptide created by genetic engineering. The animal species from which the basic fibroblast growth factor originates and the animal species from which the pluripotent stem cells in contact with the basic fibroblast growth factor originate may be the same species or different species. Preferably, they are the same species. For example, when using human-derived pluripotent stem cells, it is preferable to use Recombinant Human Fibroblast Growth Factor 2 (Cat. No. NIB 47079000) provided by Oriental Yeast Co., Ltd. as the basic fibroblast growth factor. The amount of basic fibroblast growth factor added to the culture medium is approximately 5 ng / mL to 40 ng / mL, preferably 10 ng / mL to 30 ng / mL, more preferably 15 ng / mL to 25 ng / mL, and even more preferably 18 ng / mL to 23 ng / mL, based on Recombinant Human Fibroblast Growth Factor 2 provided by Oriental Yeast Co., Ltd. The upper and lower limits of these final concentrations can be combined as appropriate. Furthermore, when using basic fibroblast growth factors other than Recombinant Human Fibroblast Growth Factor 2 provided by Oriental Yeast Co., Ltd., it is preferable to adjust the amount added so that the activity value is equivalent to that of Recombinant Human Fibroblast Growth Factor 2 provided by Oriental Yeast Co., Ltd. The activity of basic fibroblast growth factor can be evaluated by the degree of phosphorylation of ERK (MAP kinase), etc.

[0030] Bone morphogenetic factors are also called BMPs (Bone Morphogenetic Proteins). Bone morphogenetic factors may include bone morphogenetic factor 4 and bone morphogenetic factor 2. Preferably, bone morphogenetic factor 4 is used. Bone morphogenetic factors are not limited as long as they are polypeptides that have the function of bone morphogenetic factors. Preferably, recombinant bone morphogenetic factor polypeptides created by genetic recombination technology are used. The animal species from which the bone morphogenetic factors originate and the animal species from which the pluripotent stem cells that come into contact with the bone morphogenetic factors originate may be the same species or different species. Preferably, they are the same species. For example, when using human-derived pluripotent stem cells, it is preferable to use BMP-4 Bone Morphogenetic protein-4 Human Recombinant Protein, Active (PROTP12644), provided by Booster Biological Technology, as the bone morphogenetic factor. The amount of bone morphogenetic factors added to the culture medium is approximately 1 ng / mL to 20 ng / mL, preferably 5 ng / mL to 15 ng / mL, and more preferably 8 ng / mL to 12 ng / mL, based on BMP-4 provided by Booster Biological Technology. These upper and lower limits of the final concentration can be combined as appropriate. When using bone morphogenetic factors other than Booster Biological Technology's BMP-4, it is preferable to adjust the amount added so that the activity value is equivalent to that of Booster Biological Technology's BMP-4. The activity values ​​of BMP-4 and other bone morphogenetic factors can be evaluated by the degree of phosphorylation of Smad1 or Smad5, etc.

[0031] In this specification, the culture medium containing basic fibroblast growth factor and bone morphogenetic factor used to differentiate pluripotent stem cells in the first culture step is also referred to as the first differentiation induction medium.

[0032] In the first culture step, the period for culturing pluripotent stem cells in the presence of basic fibroblast growth factor and osteogenic factors is approximately 36 to 60 hours, preferably 40 to 56 hours, and more preferably 44 to 52 hours, after changing to the first differentiation induction medium. The first culture step can be carried out in a wet incubator maintained at approximately 37°C and a CO2 concentration of approximately 5%.

[0033] 1-2.Second culture step The second culture step involves culturing the cells cultured in the first culture step in the presence of basic fibroblast growth factor and osteogenic factors at a higher concentration than in the first culture step. The descriptions of the culture medium, basic fibroblast growth factor and its amount added, and osteogenic factors are the same as those described in the first culture step above.

[0034] The second culture step is characterized by contacting cells that have completed the first culture step with a higher concentration of osteogenic factors in the presence of basic fibroblast growth factor. This step makes it possible to differentiate the early mesoderm into lateral plate mesoderm (LPM).

[0035] In this specification, the culture medium containing basic fibroblast growth factor and higher concentrations of osteogenic factors, used to further differentiate cells that have completed the first culture step in the second culture step, is also referred to as the second differentiation induction medium. The second culture step is initiated after the first culture step by replacing the culture medium with the second differentiation induction medium.

[0036] The concentration of morphogenetic factors in the second differentiation induction medium is not limited as long as it is higher than the concentration of morphogenetic factors in the first differentiation induction medium. The concentration of morphogenetic factors in the second differentiation induction medium is approximately 2 to 7 times, preferably 4 to 6 times, the concentration of morphogenetic factors in the first differentiation induction medium. More preferably, the concentration of morphogenetic factors in the second differentiation induction medium is approximately 20 ng / mL to 100 ng / mL, preferably 30 ng / mL to 80 ng / mL, and more preferably 40 ng / mL to 60 ng / mL, based on BMP-4 provided by Booster Biological Technology. These upper and lower limits of the final concentration can be combined as appropriate.

[0037] In the second culture step, the period for culturing the cells in the second differentiation induction medium is approximately 36 to 60 hours, preferably 40 to 56 hours, and more preferably 44 to 52 hours, after changing to the second differentiation induction medium. The second culture process can be carried out in a wet incubator maintained at approximately 37°C and a CO2 concentration of approximately 5%.

[0038] 1-3.Third culture step In the third culture step, the cells cultured in the second culture step are cultured in the presence of a vitamin A compound, a glycogen synthase kinase-3 inhibitor, and inhibitors of activin kinase 2 and activin kinase 3. The explanation of the culture medium is referenced here.

[0039] In the third culture step, the cells cultured in the second culture step are cultured in the presence of a vitamin A compound, a glycogen synthase kinase-3 inhibitor, and inhibitors of activin kinase 2 and activin kinase 3, which makes it possible to differentiate the lateral plate mesoderm into peritoneal mesothelial cells.

[0040] In this specification, a culture medium containing a vitamin A compound, a glycogen synthase kinase-3 inhibitor, and inhibitors of activin kinase 2 and activin kinase 3, used to further differentiate cells that have completed the second culture step in the third culture step, is also referred to as the third differentiation induction medium.

[0041] The vitamin A compound is not limited as long as it has the activity of retinoic acid when taken up into cells. For example, it may include retinoic acid, retinol, retinal, retinyl ester, etc. Retinoic acid is preferred. The concentration of the vitamin A compound in the third differentiation induction medium is approximately 0.1 μM to 5 μM, preferably 0.5 μM to 3 μM, and more preferably 0.8 μM to 1.5 μM, when converted to a retinoic acid concentration.

[0042] Glycogen synthase kinase-3 is also known as GSK-3 (glycogen synthase kinase 3). There are two types of glycogen synthase kinase-3: GSK-3α, expressed from the GSK-3A gene, and GSK-3β, expressed from the GSK-3B gene. The glycogen synthase kinase-3 inhibitor used in this process is not limited as long as it inhibits both the activity of GSK-3α and the activity of GSK-3β. One inhibitor may inhibit both the activity of GSK-3α and the activity of GSK-3β. Alternatively, a mixture of inhibitors for GSK-3α and GSK-3β may be used as the glycogen synthase kinase-3 inhibitor. Preferably, the glycogen synthase kinase-3 inhibitor is one that inhibits both the activity of GSK-3α and the activity of GSK-3β, such as CHIR99021 (CAS number: 252917-06-9). The concentration of glycogen synthase kinase-3 in the differentiation induction medium is approximately 0.5 μM to 20 μM, preferably 2.5 μM to 10 μM, and more preferably 4 μM to 6 μM, when converted to CHIR99021 concentration.

[0043] Activin kinase 2 and activin kinase 3 are also called ALK2 (Activin receptor-like kinase 2) and ALK3 (Activin receptor-like kinase 3), respectively. The inhibitors used in this process to inhibit activin kinase 2 and activin kinase 3 are not limited as long as they inhibit both the activity of activin kinase 2 and the activity of activin kinase 3. One inhibitor may inhibit the activity of both activin kinase 2 and activin kinase 3. Alternatively, an inhibitor of activin kinase 2 and an inhibitor of activin kinase 3 may be used in combination as inhibitors of activin kinase 2 and activin kinase 3. Preferably, the inhibitors of activin kinase 2 and activin kinase 3 are inhibitors that inhibit the activity of both activin kinase 2 and activin kinase 3, such as LDN193189 hydrochloride (CAS number: 1062368-24-4).

[0044] The concentrations of activin kinase 2 and activin kinase 3 in the differentiation-3 induction medium are approximately 0.1 μM to 5 μM, preferably 0.5 μM to 3 μM, and more preferably 0.8 μM to 1.5 μM, when converted to LDN193189 concentrations.

[0045] In the third culture step, the period for culturing the cells in the third differentiation induction medium is approximately 5 to 7 days, preferably 120 to 168 hours, and more preferably 136 to 154 hours, after changing to the third differentiation induction medium. The third culture step can be carried out in a wet incubator maintained at approximately 37°C and a CO2 concentration of approximately 5%.

[0046] 2. Method for subculturing induced peritoneal mesothelial stem cells Induced peritoneal mesothelial stem cells (iPMCs) induced by the method described in 1. above can also be passaged. Passaging of iPMCs is not restricted as long as it is carried out under conditions that maintain the function and survival of the iPMCs. Preferably, the passage of iPMCs is carried out under conditions that maintain the function and allow for proliferation of the iPMCs.

[0047] Specifically, for example, iPMCs can be subcultured using the following method. The iPMCs induced in step 1 above are washed one to three times with a buffer such as PBS (Nacalai Tesque, Cat. No. 14249-95) or culture medium while still attached to the plate. Subsequently, accutase is added to the plate to detach the iPMCs. The reaction conditions for accutase are 15 to 40 minutes, preferably 25 to 30 minutes, at approximately 28°C for 20 minutes. The iPMCs are gently pipettered, the cell suspension is transferred to a centrifuge tube, and then centrifuged at approximately 170 g for approximately 5 minutes and collected. The supernatant is aspirated, the cells are resuspended in a culture medium such as DMEM Ham's / F12 medium containing 2% B27 supplement medium, and transferred to a plate. The cell count at subculturing is 5 × 10⁶. 4 cells / cm 2 From 1 x 10 5 cells / cm 2 It is preferable to adjust the temperature to a suitable level. The plate is preferably coated with Matrigel, iMatrix-511 silk, type I collagen, etc. For a plate coated with Matrigel, dilute Matrigel (Corning®, Cat. No. 356237) 50 times with DMEM Ham's / F12 medium and apply an amount sufficient to cover the plate, specifically 0.15 mL / cm². 2 The above amount can be added, and after incubating at room temperature or in a 37°C incubator for at least one hour, the Matrigel solution on the plate can be collected to prepare the solution. The diluted Matrigel solution can be reused. It can be reused approximately one to three times.

[0048] Furthermore, as iMatrix-511 silk-coated plates, commercially available plates such as Matrixome, Cat. No. 892021 can be used. The type I collagen coated plate is not limited as long as it is a plate coated with type I collagen derived from rats, humans, etc. As a type I collagen coated plate, commercially available products such as Corning® BioCoat® Collagen I Transparent Multiwell Plate (Cat. No. 354400) can be used. It is preferable to limit subculturing to one to two times.

[0049] 3. Induced peritoneal mesothelial stem cells This specification discloses induced peritoneal mesothelial stem cells (iPMCs) induced by the method described in 1. above, or iPMCs passaged by the method described in 2. above.

[0050] 4. Cell composition containing induced peritoneal mesothelial stem cells This specification discloses cell compositions comprising induced peritoneal mesothelial stem cells (iPMCs) induced by the method described in 1. above, or iPMCs passaged according to 2. above.

[0051] The cell composition comprises a culture medium having a composition that allows iPMCs to maintain their function as peritoneal mesothelial cells, and / or a cell preservation solution and iPMCs. The cell composition may be a cell suspension or a frozen product. Examples of cell preservation solutions include Cryoscareless (trademark) DMSO-free (Bioverde Co., Ltd.) and STEM-CELLBANKER DMSO Free GMP grade (Takara Bio Inc.). [Examples]

[0052] The present invention will be described in more detail below with reference to examples. However, the disclosure herein shall not be construed as being limited to the examples. The experiments described in these examples were conducted with the approval of the Ethics Committee of Kansai Medical University.

[0053] 1. Materials and Methods (1) Human induced pluripotent stem cells and their culture Human induced pluripotent stem cells (hiPSCs) were used as the pluripotent stem cells.

[0054] hiPSC lines (585A1) were maintained under feeder-free culture conditions. NutriStem® hPSC XF (BLG, Cat. No. 05-100-1A) was used for culturing, and the hiPSC lines were cultured at 37 °C in the presence of 5% CO2 on iMatrix-511 silk (Matrixome, Cat. No. 892021) coated plates. Colonies of hiPSCs at 70-90% confluence were excised into small squares using accutase (M&S TechnoSystems, Cat. No. AT104-100ML).

[0055] (2) Differentiation induction of human induced pluripotent stem cells Human iPSCs were divided into plates coated with Matrigel (Corning®, Cat. No. 356237) and cultured in NutriStem® medium containing 5 μM Y27632 (Focus biomolecules, Cat. No. FCS-10-2301-25) until 80% confluence was reached.

[0056] From day 0 of culture onward, human iPSCs were cultured in a serum-free differentiation medium consisting of DMEMHam's / F12 medium (Wako, Cat. No. 048-29775) and a 2% B27 supplement (Thermo Fisher Scientific, Cat. No. 17504044) as the basal culture medium. BMP4 (Boster Biological Technology, Cat. No. PROTP12644) and bFGF (Oriental Yeast, Cat. No. NIB 47079000) were added to the serum-free differentiation medium to final concentrations of 10 ng / mL and 20 ng / mL, respectively. Using this medium, pluripotent stem cells were cultured for 2 days (until day 2 of culture) at 37°C in the presence of 5% CO2 to induce early mesoderm (EM) cells. Next, the induced EM cells were cultured for 2 days (until day 4 of culture) at 37°C in the presence of 5% CO2 in serum-free differentiation medium containing 20 ng / mL of bFGF and 50 ng / mL of BMP4 at a final concentration, and LPM (lateral plate mesoderm) cells were induced. Then, the induced LPM cells were cultured for 6 days or more (until day 10 of culture) at 37°C in the presence of 5% CO2 in serum-free differentiation medium containing 5 μM CHIR99021 (Focus biomolecules, catalog no. 10-1279-25MG), 1 μM retinoic acid (LKT Labs, Inc, Cat. No. R1876), and 1 μM LDN193189 (Sigma, Cat. No. SML0559-5MG) at a final concentration, and PMC cells were induced. The culture medium was changed every two days while inducing PMCs from LPM cells.

[0057] (3) Culture of induced peritoneal mesothelial cells The induced peritoneal mesothelial cells (iPMCs) were subcultured using the following method. The iPMCs induced in 1.(2) above were washed once with PBS (Nacalai Tesque, Cat. No. 14249-95) on day 10 of culture and treated with accutase at room temperature for 30 minutes. The iPMCs were gently pipettered, the cell suspension was transferred to a centrifuge tube, and then centrifuged at 170 g for 5 minutes to collect. The supernatant was aspirated, and the cells were resuspended in DMEM Ham's / F12 medium containing 2% B27 supplement medium. The cell count was 7 × 10⁶ in the DMEM Ham's / F12 medium containing 2% B27 supplement medium. 4 cells / cm 2 The culture medium was adjusted accordingly and seeded onto a plate coated with Matrigel. The culture medium was changed during subculturing and then every two days thereafter. To prepare the Matrigel-coated plate, Matrigel (Corning®, Cat. No. 356237) was diluted 50-fold with DMEM Ham's / F12 medium and applied in an amount sufficient to completely cover the plate, specifically 0.15 mL / cm². 2 The above amount was added. After being left at room temperature or in a 37°C incubator for more than one hour, the Matrigel solution on the plate was collected. The used Matrigel solution could be reused at least three times. Similarly, iPMC was subculturized using iMatrix-511 silk-coated plates or type I collagen-coated plates (Corning® BioCoat® Collagen I 6-well transparent multiwell plate, Cat. No. 354400).

[0058] (4) RT-PCR Total RNA was extracted from cells using ISOGEN II reagent (Nippon gene, Cat. No. 311-07361). Reverse transcription was then performed using ReverTra Ace® qPCR RT Master Mix (Toyobo, Cat. No. FSQ-201). mRNA expression analysis was performed by real-time PCR using a Rotor Gene Q (Qiagen) with THUNDERBIRD® SYBR qPCR Mix (Toyobo, Cat. No. QPS-201). The sequences of the PCR primers are shown in Table 1.

[0059] [Table 1]

[0060] (5) Western blotting Lysates were collected from cells using 4× Bolt® LDS Sample Buffer (Thermo Fisher Scientific, Cat. No. B0007). Lysates were electrophoresed using 10% SDS polyacrylamide gel and blotted onto a PVDF membrane. Anti-cytokeratin 18 (R&D Systems, Cat. No. MAB7619-SP), anti-mesothelin (Santa Cruz, Cat. No. sc-33672), or anti-β actin (R&D Systems, Cat. No. MAB8929) were used as primary antibodies. Anti-mouse IgG horseradish peroxidase-linked (BETHYL, Cat. No. A90-131P) was used as the secondary antibody. Signal detection was performed using Pierce® Western Blotting Substrate (Thermo Fisher Scientific, Cat. No. NCI3106).

[0061] (6) Permeability assay iPMCs were seeded into Transwell inserts in 6-well plates (Costar, Cat. No. 3450) and cultured until confluent. 150 mg / dL urea, 3 mM KCl, or 4.0 g / dL human serum albumin and 2% B27 supplement medium as the test substance were added to DMEM Ham’s / F12 medium and added to the lower chamber. In the upper chamber, iPMCs were cultured in medium without the test substance. After 24 hours, the medium in the upper and lower chambers was collected, and the concentrations of urea nitrogen, potassium, and albumin were measured.

[0062] (7) Wound healing assay iPMCs were cultured in 12-well plates until almost confluent. Then, the cell layer in each well was scratched with a 200 μl tip to create a wound. After washing the cells in the plate, the cells were cultured in DMEM Ham’s / F12 medium supplemented with 2% B27 supplement medium. DMEM Ham’s / F12 medium supplemented with 2% B27 supplement medium with and without iPMCs was used. After staining with Hoechst33258 for 30 minutes, iPMCs were added. The number of cells to be added was 3.0×10 4 / cm 2 . The migration of cells into the wounded area was observed with a phase contrast microscope at 0, 24, and 48 hours, and the width of the wound was calculated.

[0063] 2. Results (1) Induction of peritoneal mesothelial cells iPSCs differentiated into early mesoderm by culturing for 2 days in the presence of 20 ng / mL FGF2 and 10 ng / mL BMP4 (Figure 1A). Next, they differentiated into LPM by culturing for 2 days in the presence of 20 ng / mL FGF2 and 50 ng / mL BMP4. Furthermore, LPM differentiated into peritoneal mesothelial cells by treating with 1 μM retinoic acid (RA), 5 μM CHIR99021, and 1 μM LDN193189 for at least 6 days (Figure 1A).

[0064] We monitored the differentiation progression of iPMCs using cytokeratin (CK)-18 and mesothelioma marker mesoserine (MSLN) as specific markers for PMCs, and Wilms tumor 1 (WT1) as a support marker. The results showed that CK-18 and MSLN expression peaked on day 10 of culture. On day 15, both CK-18 and MSLN expression showed a decreasing trend. On the other hand, WT1 expression increased sharply after day 10 of culture. This change, observed only in WT1, suggested the possibility that differentiation into other mesodermal organs may have occurred (Figure 1B). Furthermore, the amount of CK-18 protein showed a similar trend to the changes in mRNA expression (Figure 1C).

[0065] Furthermore, as shown in Figure 2, TBX1 and CDX2, which are markers for LPM, were elevated on day 4 of culture. This suggests that differentiation into LPM was progressing.

[0066] (2) Subculturing of iPSCs Following the protocol described in 1. above, the iPSCs reached confluence on day 10 of culture. Therefore, the iPSCs on day 10 of culture were further passaged according to the schedule shown in Figure 3A, and the cell morphology was observed, as well as the expression of the same markers as described in 2.(1) above.

[0067] Figures 3B to 3D show the results when the cells were subcultured using Matrigel-coated plates. After subculturing (cultured from day 0, the start of differentiation, to day 12), the iPMCs exhibited a monolayer appearance similar to human omentum-derived PMCs (Figure 3B). Furthermore, the expression of CK-18 and WT1 mRNA increased sharply after subculturing. On the other hand, MSLN decreased after subculturing (Figure 3C). As mentioned above, MSLN is overexpressed in tumor cells such as mesothelioma, and it was thought that the expression decreased because undifferentiated cells were eliminated during subculturing. In addition, as shown in Figure 4, the expression of the mesoderm markers TBX1 and CDX2 also decreased after subculturing. The CK-18 protein showed similar results to the mRNA (Figure 3D). Furthermore, when subculturing iPMCs was performed using iMatrix-511 silk-coated plates or type I collagen-coated plates instead of Matrigel-coated plates, the engraftment of iPMCs was also confirmed.

[0068] We attempted to increase the iPMCs by continuing to passage them, but there was almost no increase after the second passage, and no increase at all after the third passage (data not shown). Therefore, we determined that the biological function of the iPMCs should be evaluated after the second passage.

[0069] (3) Permeability assay Peritoneal dialysis utilizes the permeability of the mesothelium; therefore, the mesothelium formed from iPMCs must perform the same function as the PMCs inherent in the body. Thus, the permeability of iPMCs was evaluated using urea, KCl, and albumin. Potassium ions and urea are markers for initiating renal replacement therapy and are major substances that need to be removed from the blood of patients with renal failure. On the other hand, albumin is a major molecular substance necessary for maintaining effective circulating plasma volume and should not be removed during peritoneal dialysis. In other words, PMCs usable for peritoneal dialysis must be permeable to potassium ions and urea, but not to albumin.

[0070] An in vitro permeability assay, as shown in Figure 5A, was designed to evaluate the differences in urea, potassium, and albumin concentrations between different culture media in spaces mediated by iPMCs. The results are shown in Figures 5B and 5C. Figure 5B shows measured values, and Figure 5C shows permeability. Urea and potassium were permeated almost 100%, but albumin permeability was around 40%. From this, it was concluded that iPMCs possess the functionality to withstand peritoneal dialysis.

[0071] (4) Wound healing ability of iPMC Early healing of peritoneal injuries is crucial to preventing peritoneal adhesions. Therefore, we evaluated whether iPMCs have the ability to heal wounds. Peritoneal mesothelial cells from Passage 1 (P1) were scratched with a 200 μL tip, the wounds were observed, and the wound distance was measured. The wound distance decreased over time, and after 48 hours, the wounds were almost healed (Figure 6A, B). Furthermore, to simulate cell therapy, newly collected iPMCs from P1 were suspended in culture medium and added to the iPMCs that had undergone curettage. The added iPMCs were distinguished by staining with Hoechst. When observed 3 hours after the addition of iPMCs, Hoechst-positive iPMCs accumulated in the curettage area, and there was almost no layering and engraftment on top of the iPMCs in the uncured area. From this, it was considered that administering iPMCs may lead to wound site-specific engraftment and healing (Figure 6C). These results demonstrate that PMCs induced from pluripotent stem cells possess physiological characteristics such as permeability and healing ability, making them suitable for use in regenerative medicine.

Claims

1. A method for inducing peritoneal mesothelial cells from pluripotent stem cells, A first culture step in which pluripotent stem cells are cultured in the presence of basic fibroblast growth factor and osteogenic factors, wherein the amount of osteogenic factors added to the culture medium is 8 ng / mL to 12 ng / mL. A second culture step in which cells cultured in the first culture step are cultured in the presence of basic fibroblast growth factor and osteogenic factor, wherein the amount of osteogenic factor added to the culture medium is 40 ng / mL to 60 ng / mL. The third culture step involves culturing the cells cultured in the second culture step in the presence of a vitamin A compound; a glycogen synthase kinase-3 inhibitor; and inhibitors of activin kinase 2 and activin kinase 3. The aforementioned method.

2. The method according to claim 1, wherein the bone morphogenetic factor is bone morphogenetic factor 4.

3. The method according to claim 1 or 2, wherein the glycogen synthase kinase-3 inhibitor is CHIR99021.

4. The method according to any one of claims 1 to 3, wherein the inhibitors of activin kinase 2 and activin kinase 3 are LDN193189.

5. The culture period for the first culture step is 36 to 60 hours. The culture period for the second culture step is 36 to 60 hours. The culture period for the third culture step is 5 to 7 days. The method according to any one of claims 1 to 4.

6. The method according to any one of claims 1 to 5, wherein, when the induced peritoneal mesothelial cells are transplanted into an animal, the pluripotent stem cells are derived from the animal or from another animal that is immunocompatible with the animal.

7. The method according to any one of claims 1 to 6, wherein the pluripotent stem cells are human induced pluripotent stem cells.

8. Detach peritoneal mesothelial cells induced by the method of any one of claims 1 to 7 from a plate, Seeding the detached peritoneal mesothelial cells onto a new plate. A method for subculturing peritoneal mesothelial cells, including the method described above.

9. Peritoneal mesothelial cells induced by the method described in any one of claims 1 to 7, or passaged by the passage method described in claim 8.

10. A cell composition comprising peritoneal mesothelial cells as described in claim 9.

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