WO2020213734A1

WO2020213734A1 - Method for producing nephron progenitor cells

Info

Publication number: WO2020213734A1
Application number: PCT/JP2020/016969
Authority: WO
Inventors: 健二長船; 伸一前
Original assignee: 国立大学法人京都大学
Priority date: 2019-04-19
Filing date: 2020-04-17
Publication date: 2020-10-22
Also published as: JPWO2020213734A1; JP7471558B2

Abstract

Provided is a method for producing late presomitic mesoderm cells, the method comprising a step for culturing presomitic mesoderm cells in a medium including a fibroblast growth factor, an activator of an activin receptor kinase 4, 7, a BMP inhibitor, and a GSK3β inhibitor. In addition, provided is a method for producing posterior intermediate mesoderm cells, the method comprising a step for culturing late presomitic mesoderm cells in a medium including a fibroblast growth factor, a retinoic acid receptor agonist, and an activator of an activin receptor kinase 4, 7. There is provided a method for producing nephron organoids from pluripotent stem cells in a stepwise manner, the method being characterized by having the above-mentioned methods.

Description

Method for producing nephron progenitor cells

The present application relates to a method for producing nephron progenitor cells or nephron organoids. More specifically, it relates to a method for producing nephron progenitor cells or nephron organoids from pluripotent stem cells, particularly human pluripotent stem cells.

Currently, the number of patients with chronic kidney disease (CKD) is estimated to be about 13 million in Japan, and it is called a new national disease. There are few curative treatments for chronic kidney disease, and the number of patients with end-stage chronic renal failure who require dialysis therapy due to their progression is more than 330,000, which is a major problem not only medically but also medically economically. Kidney transplantation is one of the radical treatments for chronic kidney disease including end-stage chronic renal failure, but supply is not keeping up with demand due to a serious shortage of donor organs.

In order to develop a new cell therapy for chronic kidney disease and solve the shortage of donor organs in renal transplantation, it is necessary to establish a method for highly efficient production of renal cells and renal tissue from iPS cells.

The kidney is derived from the intermediate mesoderm, which is an early embryonic tissue. In vertebrates, the intermediate mesoderm forms three kidneys, the anterior kidney, the middle kidney, and the posterior kidney, and in mammals, the posterior kidney becomes the adult kidney. The hind kidney is caused by the interaction of two tissues, the mesenchyme and the ureteral bud. The mesenchymal is the tissue that will differentiate into the nephrons and interstitium of the adult kidney in the future, and the ureteral bud is the tissue that will differentiate into the lower renal pelvis, ureter, and part of the bladder from the collecting duct of the adult kidney. Furthermore, it has been shown that there are glomeruli constituting nephrons and nephron progenitor cells that differentiate into several types of tubular epithelial cells in the posterior renal mesenchyme (Non-Patent Documents 1 and 2).

If a method for highly efficient production of nephron progenitor cells from human iPS cells and human ES cells is established, it is considered that nephron progenitor cells produced as a source of glomeruli and renal tubules can be used for cell therapy. In the future, it is expected that the shortage of donors for kidney transplantation can be solved by reconstructing the three-dimensional kidney. Since there are many renal diseases that occur in posterior renal nephron progenitor cells, glomeruli, and renal tubules, it is also useful for creating renal disease models. Furthermore, it is expected to develop into research such as drug nephrotoxicity evaluation system and therapeutic drug development using glomeruli, renal tubules or renal tissues containing them.

Several reports have been reported on methods for inducing differentiation of Neflon progenitor cells from human iPS cells and human ES cells (Non-Patent Documents 3 to 6), but the efficiency of inducing differentiation is low because embryoid bodies are used (Non-Patent Documents). 3) Another problem was that it was unclear whether or not each stage of development was accurately reproduced (Non-Patent Documents 4 to 6). Further, although the group of the present inventors discloses a method for producing nephron progenitor cells from intermediate mesoderm cells (Patent Documents 1 and 2), a method for producing nephron progenitor cells more efficiently and stably is required. ing.

WO2014 / 200115 WO2018 / 216743

An object of the present application is to provide a method for producing nephron progenitor cells or nephron organoids from pluripotent stem cells, particularly iPS cells and ES cells. Another object of the present application is Neflon via each stage of differentiation from pluripotent stem cells to Neflon progenitor cells, anterior primitive streak cells, anterior segment mesophyll cells, late anterior segment mesophyll cells, and posterior intermediate mesoblasts. It is to provide a system for producing progenitor cells. Yet another object of the present application is each stage of differentiation from pluripotent stem cells to nephron organoids, anterior progenitor striatum cells, anterior segment mesodermal cells, late anterior segment mesodermal cells, posterior intermediate mesoderm cells, nephrone. It is to provide a system for producing nephrone organoids via progenitor cells.

This application is
(3) Late somites including the step of culturing presomites mesoderm cells in a medium containing fibroblast growth factor, activin receptor kinase activators 4 and 7, BMP inhibitors, and GSK3β inhibitors. A method for producing nodal mesoderm cells is provided.

In this embodiment, the anterior segment mesoderm cells may be cells produced from pluripotent stem cells by a method comprising the following steps (1) and (2):
(1) A step of culturing pluripotent stem cells in a medium containing activin receptor kinase kinases 4 and 7 activators and a GSK3β inhibitor to obtain anterior primordial striatal cell culture, and (2) anterior primordial. A step of culturing a striatal cell culture in a medium containing a fibroblast growth factor, a TGFβ inhibitor, a BMP inhibitor, and a GSK3β inhibitor. In addition, the presomitic mesoderm cells may be obtained by other known methods. In this embodiment, the pluripotent stem cell may be an iPS cell or a human iPS cell. That is, this aspect provides a method for producing late presomites mesoderm cells from pluripotent stem cells.

This application also
(4) Provided is a method for producing posterior intermediate mesoderm cells, which comprises a step of culturing late pre-somites mesoderm cells in a medium containing fibroblast growth factor and activin receptor kinase kinases 4 and 7 activators. To do. In this aspect, the late presomites mesoderm cells may be those produced by the method of the present application or may be obtained by other known methods. That is, this aspect provides a method for producing posterior intermediate mesoderm cells from pluripotent stem cells.

The present application also obtains posterior intermediate mesoderm cells by the method of the present application, and (5) cultures the posterior intermediate mesoderm cells in a medium containing fibroblast growth factor, GSK3β inhibitor, BMP inhibitor, and ROCK inhibitor. Provided is a method for producing Neflon progenitor cells, which comprises a step of making a nephrone precursor cell. That is, this aspect provides a method for producing nephron progenitor cells from pluripotent stem cells.

The present application also provides a method for producing a nephron organoid, which comprises the step of obtaining nephron progenitor cells by the method of the present application and further (6) culturing the nephron progenitor cells in a gas phase liquid phase interface. That is, this aspect provides a method for producing a nephron organoid from pluripotent stem cells.

The present application also provides a method for producing a nephron organoid, which comprises the step of obtaining nephron progenitor cells by the method of the present application and further (6') suspension-culturing the nephron progenitor cells.

The present application provides the method described in the claims. The method of the present application makes it possible to produce nephron progenitor cells or nephron organoids with relatively high efficiency and stability.

An example of a protocol for inducing differentiation of human pluripotent stem cells into nephron organoids (Example 1). Immunostained images of posterior intermediate mesoderm cells obtained by the method shown in FIG. 1 using antibodies against WT1 (green) and HOXD11 (red). The scale bar is 100 μm. For the nephron organoids obtained by the method shown in FIG. 1, PODOCALYXIN (glomerular podocyte marker; white, leftmost panel), LTL (Lotus tetragonolobus lectin) (proximal tubule marker; red, second from left) Panel), or immunostaining images using each antibody against E-cadherin (distal tubule marker, CDH1; green, second panel from the right), and immunostaining signals and nuclear staining with the above three antibodies. (Blue) Represents an image of merged signals (far right). An example of a protocol for inducing differentiation of human pluripotent stem cells into mesonephric duct cells. Immunostained images of GATA3 (green) and RET (red) of mesonephric duct cells induced from human iPS cells with the differentiation induction time of the first stage set to 16 or 24 hours. The scale bar is 100 μm. An example of a protocol for inducing differentiation of human pluripotent stem cells into posterior intermediate mesoderm cells when the treatment time of the first stage is 27 hours (Example 2). Immunostained image of WT1 (green) of posterior intermediate mesoderm cells induced from human iPS cell 1383D2 strain with the differentiation induction time of the first stage set to 27 hours. An example of a protocol for inducing differentiation of human pluripotent stem cells into nephron organoids in a suspension culture in the sixth stage (Example 3). A microscopic image of a nephron organoid obtained by the method described in FIG. An example of a differentiation-inducing protocol from human pluripotent stem cells to nephron organoids, in which TTNPB was removed from the fourth-stage differentiation-inducing factor (Example 4). A microscopic image of a nephron organoid obtained by the method described in FIG.

In the specification of the present application and claims, the term "approx." Accompanying a numerical value shall include up to ± 30%, ± 20%, or ± 10% of the numerical value.

In the present specification and claims, the expression "a specific type of cell" means a group of cells containing the type of cell unless otherwise specified, and the said cell group includes the specified type of cell. It may contain cells of a type other than cells. For example, "culture of a specific type of cell" means a culture of a cell group containing the cell of the specific type, and may contain cells other than the cell of the specified type. Similarly, the expression "a specific type of cell aggregate" means a cell aggregate containing the type of cell unless otherwise specified, and the cell aggregate includes cells other than the specified type of cell. Cells may be included.

As used herein and as claimed, a "pluripotent stem cell" is a stem cell that has both pluripotency and proliferative ability that can differentiate into all cells existing in the living body, for example, an embryonic stem (ES) cell. (JA Thomson et al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996)) , Biol. Reprod., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165), Embryonic stem derived from cloned embryo obtained by nuclear transplantation ( ntES) cells (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. ( 2007), Nature, 450: 497-502), sperm stem cells (“GS cells”) (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. ( 2004), Cell, 119: 1001-1012), Embryonic stem cells (“EG cells”) (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550-551), induced pluripotent stem (iPS) cells (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131 : 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); WO2007 / 069666), and cultured fibers Pluripotent cells (Muse cells) derived from germ cells or bone marrow stem cells ( WO2011 / 007900) etc. are included. In particular, mouse or human pluripotent stem cells, particularly ES cells and iPS cells, are preferably used.

The "ROCK inhibitor" is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK), for example, Y-27632 (Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya. et al., Methods Enzymol. 325,273-284 (2000)), Fasudil / HA1077 (Uenata et al., Nature 389: 990-994 (1997)), SR3677 (Feng Y et al., J Med Chem. 51: 6642) -6645 (2008)), GSK269962 (Stavenger RA et al., J Med Chem. 50: 2-5 (2007); WO2005 / 037197), H-1152 (Sasaki et al., Pharmacol. Ther. 93: 225- 232 (2002)), Wf-536 (Nakajima et al., Cancer Chemother Pharmacol. 52 (4): 319-324 (2003)) and their derivatives, as well as antisense nucleic acids and RNA interference-inducible nucleic acids against ROCK (eg, SiRNA), dominant negative variants, and their expression vectors. In addition, other known low molecular weight compounds can also be used as the ROCK inhibitor (US Patent Application Publication No. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508, the same). No. 2004/0002507, No. 2003/0125344, No. 2003/0087919, and International Publication Nos. 2003/062227, 2003/059913, 2003/062225, 2002/076976, The same No. 2004/039796). When referring to a ROCK inhibitor in the present application, one or more ROCK inhibitors may be used. The ROCK inhibitor used in the present application may preferably be Y-27632.

The "activin receptor-like kinase-4,7 activator" is a substance having an activating effect on ALK-4 and / or ALK-7, and examples thereof include activin, Nodal, and Myostatin. Preferably activin, especially activin A, is used.

"GSK3β inhibitor" is defined as a substance that inhibits the kinase activity of GSK3β protein (for example, phosphorylation ability to β-catenin), and many of them are already known, for example, BIO (also known as insilvin derivative). , GSK-3β inhibitor IX; 6-bromoinsilvin 3'-oxym), maleimide derivative SB216763 (3- (2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl)) -1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione), GSK-3β inhibitor VII (4-dibromoacetophenone), a phenylα bromomethylketone compound, L803-mts (also known as GSK3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), a cell membrane permeabilizing phosphorylating peptide, and CHIR99021 (6- [2- [4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino] ethylamino] pyridine-3 with high selectivity -Carbonitrile). These compounds are commercially available from, for example, Calbiochem, Biomol, etc., and can be easily used. The GSK3β inhibitor used in the present application may preferably be CHIR99021.

FGF1 to FGF23 are known as "fibroblast growth factors", and they may be appropriately selected from these known ones. FGF8 is preferably used in the second to fourth stages described below, and FGF9 is preferably used in the fifth stage.

A "TGFβ inhibitor" is a substance that inhibits the signal transduction from binding of TGFβ to a receptor to SMAD, and inhibits the binding of TGFβ to the ALK family of receptors, or phosphorylation of SMAD by the ALK family. As long as it is an inhibitory substance, it is not particularly limited, and for example, Lefty-1 (NCBI Accession No., mouse: NM_010094, human: NM_020997 is exemplified), SB431542, SB202190 (above, RKLindemann et al., Mol. Cancer). , 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (3- (6-methyl-2-pyridinyl)) -N-Phenyl-4- (4-quinolinyl) -1H-pyrazol-1-carbothioamide, WO2009146408), ALK5 inhibitor II (2- [3- [6-methylpyridine-2-yl] -1H-pyrazol- 4-yl] -1,5-naphthylidine), TGFβRI kinase inhibitor VIII (6- [2-tert-butyl-5- [6-methyl-pyridine-2-yl] -1H-imidazol-4-yl]- Kinoxalin) and derivatives thereof are exemplified. It can preferably be A83-01.

"BMP inhibitor" is a protein inhibitor such as Chordin, Noggin, Follistatin, Dorsomorphin 6- [4- (2-piperidin-1-yl-ethoxy) phenyl] -3-pyridin-4-yl-pyrazolo [1 , 5-a] pyrimidine, its derivatives (P. B. Yu et al. (2007), Circulation, 116: II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4: 33-41; J. Hao et al. (2008), PLoS ONE, 3 (8): e2904) and LDN193189 (4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin- 3-yl) quinoline) is exemplified. It can preferably be LDN193189.

A "retinoic acid receptor (RAR) agonist" is a naturally occurring retinoid, a chemically synthesized retinoid, a retinoic acid receptor agonist compound having no retinoid skeleton, or a natural product having retinoic acid receptor agonist activity. You can. Examples of natural retinoids that have activity as RAR agonists are retinoic acid (stereoisomeric total trans-retinoic acid (total trans RA) and 9-cis-retinoic acid (9-cis RA) are known). Can be mentioned. Chemically synthesized retinoids are known in the art (US Pat. No. 5,234,926, US Pat. No. 4,326,055, etc.). Examples of retinoic acid receptor agonist compounds that do not have a retinoid skeleton are Am80, AM580 (4-[[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl] carboxy). Amide] Benzoic Acid), TTNPB (4-[[E] -2- [5,6,7,8-Tetrahydro-5,5,8,8-Tetramethyl-2-naphthalenyl] -1-propenyl] Ben Zoic acid), AC55649 (4'-octyl- [1,1'-biphenyl] -4-carboxylic acid). Examples of natural products having retinoic acid receptor agonist activity include honokiol and magnolol (Bulletin of the Institute for Biological Function Development 9: 55-61, 2009). The RAR agonist used in the present application can be, for example, retinoic acid, AM580, TTNPB, AC55649, preferably TTNPB.

In each step of the method of the present application, the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO ₂ containing air, and the CO ₂ concentration is preferably. It is about 2 to 5%.

Before the pluripotent stem cells are provided in the method of the present application, preferably in a culture vessel coated with a pluripotent stem cell culture coating, for example, laminin coating, or commercially available, for example, iMatrix silk (Nippi Co., Ltd.). Incubate in a maintenance medium for pluripotent stem cells supplemented with a ROCK inhibitor for 12 to 48 hours, for example, about 24 hours. As the maintenance medium for pluripotent stem cells, a commercially available medium can be appropriately used, and examples thereof include StemFit ^{(registered trademark)} AK02N medium (Ajinomoto Co., Inc.). When Y-27632 is used as the ROCK inhibitor, its concentration in the medium is usually 0.1 μM to 1 mM, preferably 1 μM to 100 μM, more preferably 5 μM to 20 μM, for example about 10 μM.

Next, the differentiation of pluripotent stem cells is induced by the method of the present application. The medium can be prepared by appropriately adding factors necessary for each stage to the basal medium used for culturing animal cells. The basal medium includes, for example, MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, DMEM / F12 medium, Ham's medium. F12 medium, RPMI 1640 medium, Fischer's medium, and a mixed medium thereof are included. The basal medium may contain serum (eg, fetal bovine serum (FBS)) or may be serum-free. If necessary, for example, albumin, transferase, KnockOut Serum Replacement (KSR) (serum substitute during ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids, It may contain one or more serum substitutes such as insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non- Added to one or more substances such as essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and their equivalents, or other normal animal culture media. Can contain one or more substances.

In one embodiment, Essential 6 ^™ medium (Thermo ^), which is a serum-free medium in which L-ascorbic acid-2-phosphate magnesium, sodium selenium, insulin, LVDS ₃ and transferrin are added to DMEM / F12 medium as the basal medium. Fisher Scientific) is used to illustrate the method of the present application.

Hereinafter, each stage of the method of the present application will be described. It can be confirmed by the expression of the marker on the cell that the target cell is induced at each stage. The expression of the marker can be confirmed by a known method such as immunostaining or FACS. As the cell culture obtained at each stage, the target cell may be isolated by a known method such as FACS and used for the next stage, or the cell culture of the obtained target cell may be used as it is. It may be offered to the next stage.

First stage The first stage of the present application is a step of inducing anterior primitive streak cells from pluripotent stem cells. In this step, pluripotent stem cells are cultured in a medium containing activin receptor kinases 4 and 7 activators and GSK3 inhibitors. Further, BMP4 may be contained in the medium. In some embodiments, the process involves planar culturing of pluripotent stem cells.

When activin A is used as the activin for activin receptor kinases 4 and 7, its concentration in the medium is usually from 1 ng / ml to 10 μg / ml, preferably from 10 ng / ml to 1 μg / ml, more preferably from 50 ng / ml. 200 ng / ml, for example about 100 ng / ml.

When CHIR99021 is used as a GSK3β inhibitor, its concentration in the medium is usually 0.03 μM to 300 μM, preferably 0.3 μM to 30 μM, more preferably 1 μM to 5 μM, for example, about 3 μM.

When BMP4 is further contained, the concentration of BMP4 in the medium is usually 0.1 ng / ml to 1 μg / ml, preferably 1 ng / ml to 100 ng / ml, more preferably 5 ng / ml to 20 ng / ml, for example about 10 ng / ml. ml.

In the first stage of the present application, it is preferable to culture by exchanging only the medium on a plate for culturing pluripotent stem cells. The culturing period is preferably about 10 to 12 hours longer than the culturing time in which differentiation is preferentially induced in the anterior intermediate mesoderm under the culturing conditions of Example 2 of the present application, and is preferably 12 to 48 hours as a guide. It can be 17 to 40 hours, more preferably 20 to 38 hours, still more preferably 24 to 36 hours, 27 to 32 hours. Generation of anterior primitive streak cells can be confirmed, for example, by expression of LHX1, FOXA2, GSC, and / or BRACHYURY.

Second stage The second stage of the present application is a step of deriving anterior somites mesoderm cells from anterior primitive streak cells. The anterior primitive streak cell may be a cell obtained in the first stage, or a cell obtained by another known method may be used.

In this step, anterior primitive streak cells are cultured in a medium containing a fibroblast growth factor, a TGFβ inhibitor, a BMP inhibitor and a GSK3β inhibitor. In some embodiments, the process involves planar culturing of anterior primitive streak cells.

When FGF8 is used as the fibroblast growth factor, its concentration in the medium is usually 2 ng / ml to 20 μg / ml, preferably 20 ng / ml to 2 μg / ml, more preferably 100 ng / ml to 400 ng / ml, for example about 200 ng / ml. ml.

When A83-01 is used as a TGFβ inhibitor, its concentration in the medium is usually 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 0.5 μM to 2 μM, for example, about 1 μM.

When LDN193189 is used as a BMP inhibitor, its concentration in the medium is usually 0.001 μM to 10 μM, preferably 0.01 μM to 1 μM, more preferably 0.05 μM to 0.2 μM, for example, about 0.1 μM.

In the second stage, culturing is carried out for a total of, for example, 2 to 6 days, preferably about 3 days. For the first half of the second stage, for example, about two days, the medium of the anterior primitive streak cell culture obtained by the first stage or another method may be replaced with the medium for the second stage and cultured. In the latter half of the second stage, for example, about one day, the culture plate is transferred to an extracellular matrix protein, for example, a cell culture plate coated with laminin, for example, iMatrix silk in a commercially available product for culturing. After exchanging the cell culture plate, preferably, a ROCK inhibitor, for example, Y-27632, is further added to the medium to continue the culture. The concentration of Y-27632 in the medium may be the same as the concentration when culturing pluripotent stem cells before the start of the first stage. Generation of prosomitral mesoderm cells can be confirmed, for example, by expression of TBX6 and / or CDX2.

Third stage The third stage of the present application is a step of inducing the late anterior segment mesoderm cells from the anterior segment mesoderm cells. The anterior segment mesoderm cells may be cells obtained in the second stage, or cells obtained by another known method may be used.

In this step, anterior segment mesoderm cells are cultured in a medium containing fibroblast growth factor, activin receptor kinase activators 4, 7 activators, BMP inhibitors and GSK3β inhibitors. In some embodiments, the steps are planar culture of anterior segment mesoderm cells.

When FGF8 is used as a fibroblast growth factor, its concentration in the medium is usually 2 ng / ml to 20 μg / ml, preferably 20 ng / ml to 2 μg / ml, more preferably 100 ng / ml to 400 ng / ml, for example about 200 ng. / Ml.

When activin A is used as the activator of activin receptor kinases 4 and 7, the concentration in the medium is usually 0.1 ng / ml to 1 μg / ml, preferably 1 ng / ml to 100 ng / ml, more preferably 5 ng / ml. From ml to 20 ng / ml, for example about 10 ng / ml.

In the third stage of the present application, for example, the medium of the anterior segment mesoderm cell culture obtained in the latter half of the second stage or by another method may be replaced with the medium for the third stage and cultured. The culture period may be 12 to 48 hours, for example, about 24 hours. Generation of late presomites mesoderm cells can be confirmed, for example, by expression of TBX6, CDX2, and / or HOX11.

In the first to third stages, the concentration of the GSK3β inhibitor in the medium may be 1 to 5 μM, for example, about 3 μM. High in conventional methods for inducing differentiation of nephron progenitor cells (Taguchi A. et al., Cell Stem Cell. 2014, Takasato M. et al., Nature. 2015, and Morizane R. et al., Nat Biotechnol. 2015, etc.) Although a concentration (8 to 10 μM) of GSK3β inhibitor is used, the method of the present application can induce differentiation of nephron progenitor cells by using a relatively low concentration (1 to 5 μM) of GSK3β inhibitor. Therefore, the method of the present application has lower cytotoxicity than the conventional method, and can stably produce nephron progenitor cells from human iPS / ES cell lines.

4th stage The 4th stage is a step of inducing posterior intermediate mesoderm cells from late pre-somites mesoderm cells. The late presomites mesoderm cells may be cells obtained in the third stage, or cells obtained by another known method may be used.

In this step, late early somites mesoderm cells are cultured in a medium containing fibroblast growth factor and activin receptor kinase kinases 4 and 7 activators. In addition, the medium may contain a retinoic acid receptor agonist. In some embodiments, in this step, late presomites mesoderm cells are planarly cultured.

Further, when TTNPB is contained as a retinoic acid receptor agonist, the concentration in the medium is usually 0.001 μM to 10 μM, preferably 0.01 μM to 1 μM, more preferably 0.05 μM to 0.2 μM, for example, about 0.1 μM. is there.

In the fourth stage of the present application, for example, the medium of the late somites mesoderm cell culture obtained by the third stage or another method may be replaced with the medium for the fourth stage and cultured. The culture period may be 1 to 5 days, for example, about 3 days. Generation of posterior intermediate mesoderm cells can be confirmed, for example, by the expression of HOXD11, which indicates that it can differentiate into PAX2, SIX1, EYA1, WT1, and / or posterior kidney.

Fifth stage The fifth stage is a step of inducing the posterior intermediate mesoderm cells obtained in the fourth stage into nephron progenitor cells. In this step, posterior intermediate mesoderm cells are suspended-cultured in a medium containing a fibroblast growth factor, a GSK3β inhibitor, a BMP inhibitor, and a ROCK inhibitor.

When FGF9 is used as a fibroblast growth factor, its concentration in the medium is usually 2 ng / ml to 20 μg / ml, preferably 20 ng / ml to 2 μg / ml, more preferably 100 ng / ml to 400 ng / ml, for example about 200 ng. / Ml.

When CHIR99021 is used as a GSK3β inhibitor, its concentration in the medium is usually 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 0.5 μM to 2 μM, for example, about 1 μM.

When Y-27632 is used as the ROCK inhibitor, its concentration in the medium is usually 0.1 μM to 1 mM, preferably 1 μM to 100 μM, more preferably 5 μM to 20 μM, for example, about 10 μM.

In some embodiments, in the fifth stage, the posterior intermediate mesoderm cells obtained in the fourth stage are suspended-cultured. Suspension culture is the cultivation of cells in a culture dish in a non-adherent state. Although not particularly limited, those that have not been artificially treated (for example, coating treatment with extracellular matrix) for the purpose of improving adhesion to cells, or those that artificially suppress adhesion (for example, polyhydroxy). It can be carried out by using a material which has been coated with a polymer (Lipidure) of ethylmethacrylic acid (poly-HEMA) or 2-methacryloyloxyethyl phosphorylcholine. The culture period may be 1 to 5 days, for example, about 3 days. The production of nephron progenitor cells can be confirmed, for example, by expression of SIX2, SIX1, PAX2, and / or WT1.

6th stage The 6th stage is a step of inducing the nephron progenitor cells obtained in the 5th stage into nephron organoids. In this step, nephron progenitor cells are interfacially cultured in a gas phase liquid phase.

The medium used in this step preferably contains serum. As the serum, fetal bovine serum (FBS) may be used, and in the case of inducing differentiation of human cells, serum of another animal species such as human serum may be used. When FBS is used, its concentration in the medium is not particularly limited and may be appropriately determined by those skilled in the art. For example, it may be about 10%.

In the 6th stage, the nephron progenitor cells obtained in the 5th stage are vapor-phase liquid-phase interfacial culture. Gas-phase liquid-phase interfacial culture is carried out, for example, by culturing cells on a filter located at the interface between the gas phase and the liquid phase. The filter used in the present application is not particularly limited, but may be, for example, a polyethylene terephthalate (PET) filter (for example, a pore diameter of 8.0 μm), and as a commercial product, for example, a ThinCert cell culture insert (Greiner Bio-One) is used. it can. The culture period is not particularly limited, but is, for example, 5 days or more, preferably 7 days or more. The upper limit of the culture period is not particularly limited, but is, for example, about 3 weeks.

The production of nephron organoids can be confirmed, for example, by the expression of PODOCALYXIN, which is a glomerular podocyte marker, LTL (Lotus tetragonolobus lectin), which is a proximal tubular marker, and / or CDH1, which is a distal tubular marker. The formation of nephron organoids can be confirmed under a microscope.

6 'stage 6' stage is a step of the nephron progenitor cells obtained in the fifth stage to induce the nephron Olga maytansinoids. In this step, nephron progenitor cells are suspended and cultured.

The medium used in this step preferably contains polyvinyl alcohol. When polyvinyl alcohol is contained in the medium, excessive aggregation of nephron organoids is suppressed, and as a result, a plurality of nephron organoids having a good size can be obtained. The average molecular weight of the polyvinyl alcohol used can be 10,000 to 150,000, preferably 20,000 to 100,000, more preferably 30,000 to 70,000. The concentration of polyvinyl alcohol in the medium is usually 0.005% to 10%, preferably 0.01% to 5%, more preferably 0.05% to 2.5%, still more preferably 0.1% to 1. %.

The medium used in this step preferably contains serum and / or serum substitute. As the serum, fetal bovine serum (FBS) may be used, and in the case of inducing differentiation of human cells, serum of another animal species such as human serum may be used. As a serum substitute, KnockOut Serum Replacement (KSR) (Serum Substitute for ES Cell Culture) (Thermo Fisher Scientific) can be used. When KSR is used as a serum substitute, its concentration in the medium is not particularly limited and may be appropriately determined by those skilled in the art. For example, it may be about 10%.

In this step, the culture is preferably carried out with stirring. Stirring can be performed by rotating the culture vessel in an arc shape using, for example, a commercially available rotary shaking culture machine. The stirring speed is usually 5 to 300 rpm, preferably 10 to 150 rpm, and more preferably 30 to 100 rpm.

The culture period is not particularly limited, but is, for example, 3 days or more, preferably 5 days or more, more preferably 6 days or more, and further preferably 7 days or more. The upper limit of the culture period is not particularly limited, but is, for example, about 3 weeks.

Examples will be described below in more detail, but the present invention is not limited to the examples.

[Example 1] Induction of differentiation of nephron organoids from pluripotent stem cells (manufacturing)
Medium and differentiation-inducing factor StemFit as undifferentiated maintenance medium ^(TM) AK02N medium (Takara Bio Inc.) was used Essential 6 medium (Thermo Fisher Scientific) as a differentiation inducing medium.
100 ng / ml activin A (R & D Systems) and 3 μM CHIR99021 (Stem RD) were used as first-stage differentiation inducers.
200 ng / ml FGF8 (Peprotech), 1 μM A83-01 (Fujifilm Wako Pure Chemical Industries, Ltd.), 0.1 μM LDN193189 (Axon Medchem) and 3 μM CHIR99021 were used as the second stage differentiation inducers.
200 ng / ml FGF8, 10 ng / ml activin A, 0.1 μM LDN 193189 and 3 μM CHIR 99021 were used as the third stage differentiation inducers.
200 ng / ml FGF8, 0.1 μM TTNPB (Santa Cruz Biotechnology) and 10 ng / ml activin A were used as the fourth stage differentiation inducers.
200 ng / ml FGF9 (Peprotech), 1 μM CHIR99021, 0.1 μM LDN 193189 and 10 μM Y-27632 were used as the differentiation-inducing factors for the fifth stage.

Preparation of undifferentiated human iPS cell suspension Undifferentiated human iPS cells were cultured in 6-well plates until the cell density reached 15-20 × 10 ⁴ cells / cm ² , and 1 mL of warm 0.5 mM EDTA / PBS ( Washed twice with-). 2 mL of warm 0.5 mM EDTA / PBS (-) was added and incubated at 37 ° C. under 5% CO ₂ for 5 minutes. After incubation, EDTA / PBS (-) was removed with an ejector, and 1 mL of undifferentiated maintenance medium added so that Y-27632 was 10 μM was added. Pipetting was performed until dissociated into single cells, and an additional 2 mL of the medium was added to dilute the cell suspension. The number of cells was counted, and a cell suspension of 8 × 10 ⁴ cells / mL was prepared using an undifferentiated maintenance medium. Y-27632 was added to 10 μM to suppress apoptosis, and iMatrix-silk (Funakoshi Co., Ltd.) was added in the required amount (0.5 μL / well in the case of a 24-well plate) to adhere cells, and 2 × The cells were seeded on a 24-well plate at 10 ⁴ cells / cm ² and cultured at 37 ° C. under 5% CO ₂ for 1 day. As human iPS cells, 1383D2 strain, 1231A3 strain, and / or 1383D6 strain were used.

(1) Completely replace the differentiation-inducing medium from human iPS cells to anterior primitive streak cells, and replace with a differentiation-inducing medium (500 μL / well) containing the first-stage differentiation-inducing factor, at 37 ° C., 5% CO ₂ The cells were cultured underneath for 1 day to obtain anterior primitive streak cells.

(2) The differentiation-inducing medium from the anterior primitive streak cells to the anterior segment mesoderm cells was completely replaced with a differentiation-inducing medium (1 mL / well) containing a second-stage differentiation-inducing factor. After culturing at 37 ° C. under 5% CO ₂ for 2 days, the medium was completely replaced, and the cells were washed once with warm PBS (-). Warm Accutase (Funakoshi Co., Ltd.) was added at 300 μL / well and incubated at 37 ° C. under 5% CO ₂ for 3 minutes. Pipetting was performed until dissociation into single cells, and 5 mL / well of DMEM / F12 medium containing 5% FBS was added to stop the Accutase reaction. The number of cells was counted, and a cell suspension of 6 × 10 ⁵ cells / mL was prepared using Essential 6 medium. Add the second stage differentiation inducer and Y-27632 (10 μM) to achieve each concentration, and add 1 μL / well of iMatrix-silk to 1.5 × 10 ⁵ cells / cm ² (500 μL / well). Sown in a 24-well plate. The cells were cultured at 37 ° C. under 5% CO ₂ for 1 day to obtain presomitic mesoderm cells.

(3) The differentiation-inducing medium from the anterior segment mesoderm cells to the late anterior segment mesoderm cells was completely replaced with a differentiation-inducing medium (1 mL / well) containing a third-stage differentiation-inducing factor, and the temperature was 37 ° C. , 5% CO ₂ for 1 day to obtain late pre-somites mesoderm cells.

(4) Completely replace the differentiation-inducing medium from the late anterior segment mesoderm cells to the posterior intermediate mesoderm, and replace with the differentiation-inducing medium (1 mL / well) containing the differentiation-inducing factor of the 4th stage, at 37 ° C., 5 A posterior intermediate mesoderm was obtained by culturing under% CO ₂ for 3 days.

The obtained cells expressed both WT1 (green), which is a posterior intermediate mesoderm marker, and HOXD11 (red), which indicates that they can differentiate into the posterior kidney (Fig. 2). In each figure, the light-colored part indicates the stained part.

(5) The medium for inducing differentiation from posterior intermediate mesoderm cells to nephron progenitor cells was completely replaced, and washed once with warm PBS (-). Warm Accutase was added at 300 μL / well and incubated at 37 ° C. under 5% CO ₂ for 3 minutes. Pipetting was performed until dissociation into single cells, and 5 mL / well of DMEM / F12 medium containing 5% FBS was added to stop the Accutase reaction. The number of cells was counted, and a cell suspension of 1 × 10 ⁵ cells / mL was prepared using Essential 6 medium. Stage 5 differentiation-inducing factors were added to achieve each concentration, and 100 μL of cell suspension was seeded on V-bottom or U-bottom 96-well plates (non-adherent) (cell density 1 × 10 ⁴ cells). / Will be a well). The cells were precipitated by centrifugation (160 g, 2 minutes) to facilitate the formation of cell clumps. The cells were cultured at 37 ° C. under 5% CO ₂ for 3 days to obtain a cell mass of nephron progenitor cells.

(6) Induction of Differentiation from Nephron Progenitor Cells to Nephron Organoids The cell mass was placed on a filter and cultured at the vapor-liquid phase interface for 7 days or longer to obtain nephron organoids. The medium used was Essential 6 plus 10% FBS.

Regarding the obtained nephron organoids, PODOCALYXIN (glomerular podocyte marker; white), LTL (Lotus tetragonolobus lectin) (proximal tubule marker; red), E-cadherin (distal tubule marker, CDH1; green) Immunostaining and nuclear staining (blue) using each antibody were performed, and the obtained image is shown in FIG. Many PODOCALYXIN-positive (white) dot-shaped signals were observed in the organoid, and many LTL signals (pale color on the black-and-white image) were observed at the site adjacent to the signal. Furthermore, many E-cadherin signals (pale on the black-and-white image) were observed not only at the same site as the LTL signal but also at the center of the organoid.

From this result, from the surface to the center of the organoid, a series of PODOCALYXIN-positive cell groups (glomerular podocytes), LTL-positive structures (proximal tubules), and E-CADHERIN-positive structures (distal tubules) were continuous. It became clear that a nephron structure was generated. Therefore, it was shown that the posterior intermediate mesoderm prepared in this example has the ability to differentiate into nephrons.

[Example 2] Examination of first stage differentiation induction period When pluripotent stem cells are sequentially cultured in the presence of the factors shown in FIG. 4, they differentiate predominantly into the anterior intermediate mesoderm and further differentiate into the Wolff's duct. It is known to do (Costantini F. & Kopan R., Dev Cell. 18 (5). 2010). Normally, nephron progenitor cells do not arise from the anterior intermediate mesoderm, so if the proportion of cells towards the anterior intermediate mesoderm (from pluripotent stem cells) can be reduced, the proportion of cells that become the posterior intermediate mesoderm is relative. It can be expected that more nephron progenitor cells or nephron organoids will be obtained as a result. Therefore, under the culture conditions of FIG. 4, the conditions under which the proportion of cells from human iPS cells toward the anterior intermediate mesoderm decreased by changing the culture period of the first stage were examined. After the completion of the third stage, the expression of each marker protein in the Wolff tube (GATA) and the tip of the Wolff tube (RET) was analyzed by immunostaining.

As a result, as shown in FIG. 5, it was found that the number of GATA3 positive cells increased significantly when the first stage was 16 hours (left figure), but the number of GATA3 positive cells decreased sharply at 24 hours. (Right figure). Therefore, it was clarified that the number of cells that differentiate into the anterior intermediate mesoderm decreases when the culture period of the first stage is long.

Based on this result, we examined the culture period of the first stage in which more pluripotent stem cells can be induced to differentiate into the posterior intermediate mesoderm. Human iPS cells were cultured under the conditions shown in FIG. 6, and immunostained with an anti-WT-1 antibody after the completion of the fourth stage. As a result, it was clarified that the number of WT-1-positive cells increased significantly when the cells were cultured for about 10 to 12 hours longer than the culture time (about 16 hours) in which differentiation was preferentially induced in the anterior intermediate mesoderm. (Fig. 7). In addition, as a result of examining a plurality of human iPS cells, the culture period of the first stage in which the proportion of cells that became the posterior intermediate mesoderm was the highest was about 27 hours and about 32 hours for the 1383D2 strain and the 1231A3 strain, respectively. ..

Therefore, if the culture period of the first stage is 17 to 40 hours, more preferably 20 to 38 hours, and further preferably 24 to 36 hours, more human pluripotent stem cells can be induced to differentiate into the posterior intermediate mesoderm. I understood.

[Example 3] Examination of conditions for inducing differentiation of nephron precursor cells into nephron organoids For the step of differentiation of nephron precursors into nephron organoids, a simpler culture method (not only gas-phase liquid phase interfacial culture) was attempted. It was. Human iPS cells were cultured under the conditions shown in FIG. 8, and the presence or absence of nephron organoid was observed 10 days after the start of the culture. Specifically, up to step (5), the cells were cultured under the same conditions as in [Example 1], and in step (6), the nephron progenitor cell mass contained 0.5% polyvinyl alcohol (PVA) and 10% KSR. Suspension culture (shaking at 50 rpm using a shaker on a low-adhesion 35 mm dish) was performed in Essential 6. Two types of PVA with different molecular weights (MW: 30,000-70,000 and 85,000-124,000) were used.

As shown in FIG. 9, a large number of nephron organoids having almost the same size were obtained when any PVA was used. When PVA with a high molecular weight (MW: 85,000-124,000, right image in Fig. 9) was used, PVA fibers tended to become filamentous and entangled with organoids, so PVA with a not too high molecular weight (for example, MW). : 30,000-70,000 (Fig. 9, left image) seems to be preferable.

Therefore, it was clarified that the nephron progenitor cells obtained by the method according to the present invention can be induced to differentiate into nephron organoids by suspension culture. Furthermore, at that time, it was clarified that when a PVA-containing medium was used, excessive aggregation was suppressed and a plurality of nephron organoids having almost the same size were obtained.

[Example 4] (4) Examination of conditions for inducing differentiation of late early somites mesoderm cells into posterior intermediate mesoderm Human iPS cells were cultured under the conditions shown in FIG. 10 to induce differentiation into nephron organoids. .. The condition is that the retinoic acid receptor agonist is removed from the medium of step (4) in the culture condition (FIG. 1) of [Example 1].
As shown in FIG. 11, nephron organoids were also obtained when cultured under the conditions of FIG.
Therefore, (4) a retinoic acid receptor agonist is not essential for inducing differentiation of late presomites mesoderm cells into posterior middle mesoderm, and fibroblast growth factor and activin receptor kinase kinases 4 and 7 activators are used. It was shown that if there is, differentiation can be induced.

Claims

(3) Late somites including the step of culturing presomites mesoderm cells in a medium containing fibroblast growth factor, activin receptor kinase activators 4 and 7, BMP inhibitors, and GSK3β inhibitors. Method for producing node mesoderm cells.
The method according to claim 1, wherein the fibroblast growth factor is FGF8, the activin of activin receptor kinases 4 and 7 is activin A, the BMP inhibitor is LDN193189, and the GSK3β inhibitor is CHIR99021.
The method according to claim 1 or 2, wherein the anterior segment mesoderm cells are cells produced from pluripotent stem cells by a method comprising the following steps (1) and (2):
(1) A step of culturing pluripotent stem cells in a medium containing activin receptor kinase kinases 4 and 7 activators and a GSK3β inhibitor to obtain anterior primordial striatal cell culture, and (2) anterior primordial. A step of culturing a striatal cell culture in a medium containing a fibroblast growth factor, a TGFβ inhibitor, a BMP inhibitor, and a GSK3β inhibitor.
In steps (1) and (2), the activin of activin receptor kinases 4 and 7 is activin A, the GSK3β inhibitor is CHIR99021, the fibroblast growth factor is FGF8, and the TGFβ inhibitor is. The method of claim 3, wherein A83-01 and the BMP inhibitor is LDN193189.
The method according to claim 3 or 4, wherein in the steps (1) to (3), the concentration of the GSK3β inhibitor is 1 to 5 μM.
The method according to any one of claims 3 to 5, wherein the pluripotent stem cell is an iPS cell.
The method according to claim 6, wherein the iPS cells are human iPS cells.
The late presomites mesoderm cells are obtained by the method according to any one of claims 1 to 7, and (4) the late presomites mesoderm cells are obtained from fibroblast growth factor and activin receptor kinase 4,7. A method for producing posterior intermediate mesoderm cells, which comprises a step of culturing in a medium containing an activator of.
(4) A method for producing posterior intermediate mesoderm cells, which comprises a step of culturing late pre-somites mesoderm cells in a medium containing fibroblast growth factor and activin receptor kinase kinases 4 and 7.
The method according to claim 8 or 9, wherein in the step (4), the fibroblast growth factor is FGF8 and the activin of activin receptor kinases 4 and 7 is activin A.
The method according to any one of claims 8 to 10, wherein in the step (4), the medium further contains a retinoic acid receptor agonist.
The method according to claim 11, wherein in the step (4), the retinoic acid receptor agonist is TTNPB.
The posterior intermediate mesoderm cells are obtained by the method according to any one of claims 8 to 12, and (5) the posterior intermediate mesoderm cells are obtained from fibroblast growth factor, GSK3β inhibitor, BMP inhibitor, and ROCK inhibitor. A method for producing nephron progenitor cells, which comprises a step of culturing in a medium containing.
The method according to claim 13, wherein in the step (5), the fibroblast growth factor is FGF9, the GSK3β inhibitor is CHIR99021, the BMP inhibitor is LDN193189, and the ROCK inhibitor is Y-27632.
A method for producing a nephron organoid, which comprises the step of obtaining nephron progenitor cells by the method according to any one of claims 13 or 14, and further (6) culturing the nephron progenitor cells in a gas phase liquid phase interface.
A method for producing a nephron organoid, which comprises the step of obtaining nephron progenitor cells by the method according to any one of claims 13 or 14, and further (6') suspension-culturing the nephron progenitor cells.
The method according to claim 16, wherein the step (6') is a step of suspension culturing in a medium containing polyvinyl alcohol.