JP7471558B2 - Method for producing nephron progenitor cells - Google Patents

Description

The present application relates to a method for producing nephron progenitor cells or nephron organoids. More particularly, the present application 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) in Japan is estimated at approximately 13 million, and it has been called the new national disease. There are few curative treatments for chronic kidney disease, and as the disease progresses, there are more than 330,000 patients with end-stage chronic renal failure who require dialysis therapy, posing a major problem not only medically but also medical economically. Kidney transplantation is one of the radical treatments for chronic kidney disease, including end-stage chronic renal failure, but due to a serious shortage of donor organs, supply is not keeping up with demand at all.

In order to develop new cell therapies for chronic kidney disease and to resolve the shortage of donor organs for kidney transplants, it is necessary to establish methods for producing kidney cells and kidney tissue from iPS cells with high efficiency.

The kidney is derived from the intermediate mesoderm, an early embryonic tissue. In vertebrates, three kidneys, the pronephros, mesonephros, and metanephros, are formed from the intermediate mesoderm, and in mammals, the metanephros becomes the adult kidney. The metanephros develops through the interaction of two tissues, the mesenchyme and the ureteric bud. The mesenchyme is the tissue that will differentiate into the nephrons and interstitium of the adult kidney, and the ureteric bud is the tissue that will differentiate from the collecting duct into the lower renal pelvis, ureter, and part of the bladder of the adult kidney. Furthermore, it has been shown that the metanephric mesenchyme contains nephron progenitor cells that differentiate into the glomeruli that make up the nephrons and several types of renal tubular epithelial cells (Non-Patent Documents 1 and 2).

If a method for producing nephron progenitor cells from human iPS cells or human ES cells with high efficiency can be established, it is believed that the produced nephron progenitor cells could be used as a source of glomeruli and tubules in cell therapy. It is also expected that in the future, the shortage of donors for kidney transplants can be resolved by reconstructing three-dimensional kidneys. Since many kidney diseases occur in metanephric nephron progenitor cells, glomeruli, and tubules, this method is also useful for creating kidney disease models. Furthermore, it is expected that research on drug nephrotoxicity evaluation systems and drug development using glomeruli, tubules, or kidney tissues containing them will progress.

Several methods have been reported for inducing differentiation of nephron progenitor cells from human iPS cells or human ES cells (Non-Patent Documents 3-6), but the efficiency of differentiation induction is low because embryoid bodies are used (Non-Patent Document 3), and it is unclear whether each stage of development is accurately reproduced (Non-Patent Documents 4-6). In addition, the inventors' group has disclosed a method for producing nephron progenitor cells from intermediate mesoderm cells (Patent Documents 1 and 2), but there is a need for a method for producing nephron progenitor cells more efficiently and stably.

WO2014/200115 WO2018/216743

Osafune K., et al., Development 2006;133:151-161. Kobayashi A., et al., Cell Stem Cell 2008;3:169-181. Taguchi A., et al., Cell Stem Cell. 2014;14:53-67. Takasato M., et al., Nat Cell Biol, 2014;16:118-126. Takasato M., et al., Nature, 2015; 526: 564-568. Morizane R., et al., Nat Biotechnol, 2015;33:1193-1200.

The present application aims to provide a method for producing nephron progenitor cells or nephron organoids from pluripotent stem cells, particularly iPS cells or ES cells. Another object of the present application is to provide a system for producing nephron progenitor cells through the various differentiation stages from pluripotent stem cells to nephron progenitor cells, anterior primitive streak cells, presomitic mesoderm cells, late presomitic mesoderm cells, and posterior intermediate mesoderm cells. Yet another object of the present application is to provide a system for producing nephron organoids through the various differentiation stages from pluripotent stem cells to nephron organoids, anterior primitive streak cells, presomitic mesoderm cells, late presomitic mesoderm cells, posterior intermediate mesoderm cells, and nephron progenitor cells.

The present application,
(3) Provided is a method for producing late presomitic mesoderm cells, comprising the step of culturing presomitic mesoderm cells in a medium containing a fibroblast growth factor, an activator of activin receptor kinase 4, 7, a BMP inhibitor, and a GSK3β inhibitor.

In this embodiment, the presomitic mesoderm cells may be cells produced from pluripotent stem cells by a method comprising the following steps (1) and (2):
(1) culturing pluripotent stem cells in a medium containing an activator of activin receptor kinase 4, 7 and a GSK3β inhibitor to obtain an anterior primitive streak cell culture; and (2) culturing the anterior primitive streak cell culture in a medium containing fibroblast growth factor, a TGFβ inhibitor, a BMP inhibitor and a GSK3β inhibitor. The presomitic mesoderm cells may also be obtained by other known methods. In this embodiment, the pluripotent stem cells may be iPS cells or human iPS cells. That is, this embodiment provides a method for producing late presomitic mesoderm cells from pluripotent stem cells.

The present application also provides:
(4) A method for producing posterior intermediate mesoderm cells is provided, comprising the step of culturing late pre-somitic mesoderm cells in a medium containing a fibroblast growth factor and an activator of activin receptor kinase 4, 7. In this embodiment, the late pre-somitic mesoderm cells may be produced by the method of the present application or may be obtained by other known methods. That is, this embodiment provides a method for producing posterior intermediate mesoderm cells from pluripotent stem cells.

The present application also provides a method for producing nephron progenitor cells, comprising the steps of obtaining posterior intermediate mesoderm cells by the method of the present application, and (5) culturing the posterior intermediate mesoderm cells in a medium containing a fibroblast growth factor, a GSK3β inhibitor, a BMP inhibitor, and a ROCK inhibitor. That is, this embodiment provides a method for producing nephron progenitor cells from pluripotent stem cells.

The present application also provides a method for producing a nephron organoid, comprising obtaining nephron progenitor cells by the method of the present application, and further comprising the step of (6) culturing the nephron progenitor cells at an air-liquid interface. That is, the present embodiment provides a method for producing a nephron organoid from pluripotent stem cells.

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

The present application provides a method as 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 from human pluripotent stem cells into nephron organoids (Example 1). Immunostaining images of posterior intermediate mesoderm cells obtained by the method described in Figure 1, using antibodies against WT1 (green) and HOXD11 (red). The scale bar is 100 μm. Nephron organoids obtained by the method described in Figure 1 were immunostained using antibodies against PODOCALYXIN (a marker for glomerular podocytes; white, the leftmost panel), LTL (Lotus tetragonolobus lectin) (a marker for proximal tubules; red, the second panel from the left), or E-cadherin (a marker for distal tubules, CDH1; green, the second panel from the right), as well as an image (the rightmost panel) in which the immunostaining signals from the above three antibodies and the nuclear staining (blue) signals were merged. An example of a protocol for inducing differentiation of human pluripotent stem cells into mesonephric duct cells. Immunostaining of mesonephric duct cells induced from human iPS cells with the first stage differentiation induction time of 16 or 24 hours for GATA3 (green) and RET (red). Scale bar, 100 μm. 1 shows an example of a protocol for inducing differentiation of human pluripotent stem cells into posterior intermediate mesoderm cells when the first stage treatment time is 27 hours (Example 2). Immunostained image of WT1 (green) in posterior intermediate mesoderm cells induced from human iPS cell line 1383D2 after 27 hours of differentiation induction at the first stage. An example of a protocol for inducing differentiation from human pluripotent stem cells to nephron organoids, in which stage 6 is performed using suspension culture (Example 3). Microscopic images of nephron organoids obtained by the method described in FIG. An example of a differentiation induction protocol from human pluripotent stem cells to nephron organoids, in which TTNPB is excluded from the differentiation induction factor at the fourth stage (Example 4). Microscopic images of nephron organoids obtained by the method described in FIG. 10.

In this specification and claims, when the word "approximately" is used in conjunction with a numerical value, it is intended to include values up to ±30%, ±20%, or ±10% of the numerical value.

In the present specification and claims, unless otherwise specified, the expression "a certain type of cell" means a cell group containing the said type of cell, and the said cell group may contain cells of types other than the specified type of cell. For example, "a culture of a certain type of cell" means a culture of a cell group containing the said type of cell, and may contain cells of types other than the specified type of cell. Similarly, the expression "a certain type of cell aggregate" means a cell aggregate containing the said type of cell, and the said cell aggregate may contain cells of types other than the specified type of cell, unless otherwise specified.

In the present specification and claims, the term "pluripotent stem cells" refers to stem cells that have both pluripotency and proliferation ability and can differentiate into all cells present in the body, and includes, for example, embryonic stem (ES) cells (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165), cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer (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), spermatogonial 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 germ cells ("EG cells") (Y. Matsui et al. (1992), Cell, 70:841-847; J.L. 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 pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells (WO2011/007900). 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), and examples thereof include 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 against ROCK, RNA interference-inducing nucleic acids (e.g., siRNAs), dominant negative mutants, and expression vectors thereof. Other known low molecular weight compounds can also be used as ROCK inhibitors (U.S. Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508, 2004/0002507, 2003/0125344, 2003/0087919, and International Publication Nos. 2003/062227, 2003/059913, 2003/062225, 2002/076976, and 2004/039796). When referring to ROCK inhibitors in this application, one or more ROCK inhibitors may be used. The ROCK inhibitor used in this application may preferably be Y-27632.

"Activin receptor-like kinase-4, 7 activators" are substances that have an activating effect on ALK-4 and/or ALK-7, such as activin, nodal, and myostatin. Activin, particularly activin A, is preferably used.

"GSK3β inhibitors" are defined as substances that inhibit the kinase activity of GSK3β protein (e.g., the ability to phosphorylate β-catenin), and many of them are already known. For example, the indirubin derivative BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin 3'-oxime), the maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), and SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2 -nitrophenyl)-1H-pyrrole-2,5-dione), GSK-3β inhibitor VII (4-dibromoacetophenone), a phenyl α bromomethyl ketone compound, L803-mts (also known as GSK3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2), a cell membrane permeable phosphorylated peptide, and CHIR99021 (6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile), which has high selectivity. These compounds are commercially available from, for example, Calbiochem and Biomol, 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 an appropriate factor may be selected from these known factors. FGF8 is preferably used in the second to fourth stages described below, and FGF9 is preferably used in the fifth stage.

"TGFβ inhibitors" are substances that inhibit signal transduction from the binding of TGFβ to the receptor to SMAD, and are not particularly limited as long as they are substances that inhibit the binding to the receptor ALK family, or substances that inhibit the phosphorylation of SMAD by the ALK family. Examples of such substances include Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997), SB431542, SB202190 (R.K.Lindemann 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-pyrazole-1-carbothioamide, WO2009146408), ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline) and derivatives thereof. A83-01 may be preferable.

Examples of "BMP inhibitors" include protein inhibitors such as Chordin, Noggin, and 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; P.B. 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). Preferably, it may be LDN193189.

A "retinoic acid receptor (RAR) agonist" may be a naturally occurring retinoid, a chemically synthesized retinoid, a retinoic acid receptor agonist compound that does not have a retinoid backbone, or a natural product that has retinoic acid receptor agonist activity. An example of a naturally occurring retinoid with RAR agonist activity is retinoic acid, the stereoisomers of which are all-trans-retinoic acid (all-trans-RA) and 9-cis-retinoic acid (9-cis-RA). Chemically synthesized retinoids are known in the art (e.g., U.S. Pat. No. 5,234,926; U.S. Pat. No. 4,326,055). Examples of retinoic acid receptor agonist compounds that do not have a retinoid skeleton include Am80, AM580 (4-[[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]carboxamido]benzoic acid), TTNPB (4-[[E]-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl]benzoic acid), and AC55649 (4'-octyl-[1,1'-biphenyl]-4-carboxylic acid). Examples of natural products that have retinoic acid receptor agonist activity include honokiol and magnolol (Bulletin of the Institute of Biological Functions Research 9: 55-61, 2009). The RAR agonist used in the present application may be, for example, retinoic acid, AM580, TTNPB, AC55649, preferably TTNPB.

In each step of the method of the present application, the culture temperature is, but not limited to, about 30 to 40°C, preferably about 37°C, and the culture is performed in an atmosphere of _CO2- containing air, with the _CO2 concentration being preferably about 2 to 5%.

Prior to being subjected to the method of the present application, the pluripotent stem cells are preferably cultured in a culture vessel coated with a coating for pluripotent stem cell culture, such as a laminin coating, for example a commercially available coating such as iMatrix silk (Nippi Corporation), in a pluripotent stem cell maintenance medium containing a ROCK inhibitor for 12 to 48 hours, for example about 24 hours. As the pluripotent stem cell maintenance medium, a commercially available medium can be appropriately used, for example StemFit ^{(registered trademark)} AK02N medium (Ajinomoto Co., Inc.). When Y-27632 is used as the ROCK inhibitor, the 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 the pluripotent stem cells is induced by the method of the present application. The medium can be prepared by appropriately adding factors required for each stage to a basal medium used for culturing animal cells. Examples of basal media include 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 F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If desired, the medium may contain one or more serum substitutes, such as, for example, albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like, or one or more substances typically added to animal culture media.

In one embodiment, the method of the present application is carried out using, as a basal medium, Essential 6 ^(trademark) medium (Thermo Fisher Scientific), which is a serum-free medium containing DMEM/F12 medium supplemented with L-ascorbic acid-2-phosphate magnesium, sodium selenium, insulin, _NaHCO3 , and transferrin.

Each stage of the method of the present application is described below. In each stage, induction of the target cells can be confirmed by the expression of a marker on the cells. The expression of the marker can be confirmed by known methods such as immunostaining or FACS. From the cell culture obtained at each stage, the target cells may be isolated by known methods such as FACS and used in the next stage, or the obtained cell culture of the target cells may be directly subjected 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, the pluripotent stem cells are cultured in a medium containing an activator of activin receptor kinase 4, 7 and a GSK3 inhibitor. The medium may further contain BMP4. In one embodiment, in this step, the pluripotent stem cells are plate-cultured.

When activin A is used as an activator of activin receptor kinase 4, 7, its concentration in the culture medium is typically 1 ng/ml to 10 μg/ml, preferably 10 ng/ml to 1 μg/ml, more preferably 50 ng/ml to 200 ng/ml, for example about 100 ng/ml.

When CHIR99021 is used as a GSK3β inhibitor, its concentration in the culture medium is typically 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 included, the concentration of BMP4 in the culture medium is typically 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.

In the first stage of the present application, the cells may be cultured preferably in a plate for pluripotent stem cell culture with only the medium replaced. The culture period is preferably about 10 to 12 hours longer than the culture period in which differentiation is preferentially induced into anterior intermediate mesoderm under the culture conditions of Example 2 of the present application, and may be, as a guideline, 12 to 48 hours, preferably 17 to 40 hours, more preferably 20 to 38 hours, and even more preferably 24 to 36 hours or 27 to 32 hours. The generation of anterior primitive streak cells can be confirmed by, for example, the expression of LHX1, FOXA2, GSC, and/or BRACHYURY.

Second Stage The second stage of the present invention is a step of inducing presomitic mesoderm cells from anterior primitive streak cells. The anterior primitive streak cells may be the cells obtained in the first stage, or cells obtained by another known method may be used.

In this step, the 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 one embodiment, in this step, the anterior primitive streak cells are plate-cultured.

When FGF8 is used as a fibroblast growth factor, its concentration in the culture medium is typically 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 A83-01 is used as a TGFβ inhibitor, its concentration in the culture medium is typically 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 culture medium is typically 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.

When CHIR99021 is used as a GSK3β inhibitor, its concentration in the culture medium is typically 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.

In the second stage, the cells are cultured for a total of, for example, 2 to 6 days, preferably about 3 days. During the first half of the second stage, for example, about 2 days, the medium of the anterior primitive streak cell culture obtained in the first stage or by another method may be replaced with the medium for the second stage and cultured. During the second half of the second stage, for example, about 1 day, the culture plate is transferred to a cell culture plate coated with an extracellular matrix protein, for example, laminin, for example, commercially available iMatrix silk, and cultured. After replacing the cell culture plate, it is preferable to further add a ROCK inhibitor, for example, Y-27632, to the medium and continue the culture. The concentration of Y-27632 in the medium may be the same as that used when culturing pluripotent stem cells before the start of the first stage. The generation of presomitic mesoderm cells can be confirmed, for example, by the expression of TBX6 and/or CDX2.

Stage 3 The third stage of the present invention is a step of inducing late presomitic mesoderm cells from presomitic mesoderm cells. The presomitic mesoderm cells may be the cells obtained in Stage 2, or cells obtained by another known method.

In this step, the presomitic mesoderm cells are cultured in a medium containing a fibroblast growth factor, an activator of activin receptor kinase 4, 7, a BMP inhibitor, and a GSK3β inhibitor. In one embodiment, in this step, the presomitic mesoderm cells are plate-cultured.

When FGF8 is used as a fibroblast growth factor, its concentration in the culture medium is typically 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 an activator of activin receptor kinase 4, 7, its concentration in the culture medium is typically 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.

When LDN193189 is used as a BMP inhibitor, its concentration in the culture medium is typically 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.

When CHIR99021 is used as a GSK3β inhibitor, its concentration in the culture medium is typically 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.

In the third stage of the present application, for example, the medium of the presomitic mesoderm cell culture obtained in the latter half of the second stage or by other methods may be replaced with a third stage medium and cultured. The culture period may be 12 to 48 hours, for example, about 24 hours. The generation of late presomitic mesoderm cells may be confirmed by, for example, the 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. Conventional methods for inducing differentiation of nephron progenitor cells (e.g., Taguchi A. et al., Cell Stem Cell. 2014, Takasato M. et al., Nature. 2015, and Morizane R. et al., Nat Biotechnol. 2015) use high concentrations (8 to 10 μM) of GSK3β inhibitors, but the method of the present application can induce differentiation of nephron progenitor cells using relatively low concentrations (1 to 5 μM) of GSK3β inhibitors. Therefore, the method of the present application has lower cytotoxicity than conventional methods, and can stably produce nephron progenitor cells from human iPS/ES cell lines.

Stage 4 Stage 4 is a step of inducing posterior intermediate mesoderm cells from late presomitic mesoderm cells. The late presomitic mesoderm cells may be cells obtained in Stage 3 or cells obtained by another known method.

In this step, the late presomitic mesoderm cells are cultured in a medium containing fibroblast growth factor and an activator of activin receptor kinase 4, 7. The medium may further contain a retinoic acid receptor agonist. In one embodiment, in this step, the late presomitic mesoderm cells are plate-cultured.

When FGF8 is used as a fibroblast growth factor, its concentration in the culture medium is typically 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 an activator of activin receptor kinase 4, 7, its concentration in the culture medium is typically 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.

Furthermore, when TTNPB is included as a retinoic acid receptor agonist, its concentration in the culture medium is typically 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 fourth stage of the present application, for example, the medium of the late presomitic mesoderm cell culture obtained in the third stage or by other methods 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. The generation of posterior intermediate mesoderm cells may be confirmed by the expression of, for example, PAX2, SIX1, EYA1, WT1, and/or HOXD11, which indicates that the cells can differentiate into metanephros.

Stage 5 Stage 5 is a step of inducing the posterior intermediate mesoderm cells obtained in Stage 4 into nephron progenitor cells. In this step, the posterior intermediate mesoderm cells are cultured in suspension 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 culture medium is typically 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 culture medium is typically 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 culture medium is typically 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.

When Y-27632 is used as a ROCK inhibitor, its concentration in the culture medium is typically 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 one embodiment, in the fifth stage, the posterior intermediate mesoderm cells obtained in the fourth stage are cultured in suspension. Suspension culture refers to culturing cells in a non-adherent state to a culture dish. Although not particularly limited, cells that have not been artificially treated (e.g., coated with extracellular matrix, etc.) to improve adhesion to the cells, or cells that have been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA) or 2-methacryloyloxyethyl phosphorylcholine polymer (Lipidure)) may be used. The culture period may be 1 to 5 days, for example, about 3 days. The generation of nephron progenitor cells can be confirmed, for example, by the expression of SIX2, SIX1, PAX2, and/or WT1.

Stage 6 Stage 6 is a step of inducing the nephron progenitor cells obtained in Stage 5 into nephron organoids. In this step, the nephron progenitor cells are cultured at the air-liquid interface.

The medium used in this step preferably contains serum. As serum, fetal bovine serum (FBS) may be used, or serum from other animal species may be used, for example, human serum in the case of differentiation induction of human cells. When FBS is used, its concentration in the medium is not particularly limited and may be appropriately determined by a person skilled in the art. For example, it may be about 10%.

In the sixth stage, the nephron progenitor cells obtained in the fifth stage are cultured at the gas-liquid interface. The gas-liquid interface culture is performed, for example, by culturing cells on a filter located at the interface between the gas and liquid phases. The filter used in the present application is not particularly limited, but may be, for example, a polyethylene terephthalate (PET) filter (e.g., pore size 8.0 μm), and a commercially available product such as ThinCert cell culture insert (Greiner Bio-One) can be used. The culture period is not particularly limited, but may be, for example, 5 days or more, preferably 7 days or more. The upper limit of the culture period is not particularly limited, but may be, for example, about 3 weeks.

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

Stage 6' Stage 6' is a step of inducing the nephron progenitor cells obtained in Stage 5 into nephron organoids. In this step, the nephron progenitor cells are cultured in suspension.

The medium used in this step preferably contains polyvinyl alcohol. When the medium contains polyvinyl alcohol, excessive aggregation of nephron organoids is suppressed, and as a result, multiple nephron organoids with well-balanced sizes can be obtained. The average molecular weight of the polyvinyl alcohol used may be 10,000 to 150,000, preferably 20,000 to 100,000, and 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%, and even more preferably 0.1% to 1%.

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

In this step, the culture is preferably performed with stirring. The stirring can be performed, for example, by rotating the culture vessel in an arc using a commercially available rotary shaker 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 even 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 present invention will be explained in further detail below with reference to examples, but the present invention is not limited to these examples in any way.

[Example 1] Differentiation induction of nephron organoids from pluripotent stem cells (production)
Culture medium and differentiation factors
StemFit ^{(registered trademark)} AK02N medium (Takara Bio Inc.) was used as the undifferentiated state maintenance medium, and Essential 6 medium (Thermo Fisher Scientific) was used as the differentiation induction medium.
As differentiation inducers for the first stage, 100 ng/ml activin A (R&D Systems) and 3 μM CHIR99021 (Stem RD) were used.
The second stage differentiation inducers used were 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.
As differentiation inducers for the third stage, 200 ng/ml FGF8, 10 ng/ml activin A, 0.1 μM LDN193189 and 3 μM CHIR99021 were used.
As differentiation inducers for the fourth stage, 200 ng/ml FGF8, 0.1 μM TTNPB (Santa Cruz Biotechnology), and 10 ng/ml activin A were used.
As differentiation inducers for the fifth stage, 200 ng/ml FGF9 (Peprotech), 1 μM CHIR99021, 0.1 μM LDN193189 and 10 μM Y-27632 were used.

Preparation of undifferentiated human iPS cell suspension Undifferentiated human iPS cells were cultured in a 6-well plate until the cell density reached 15-20×10 ⁴ cells/cm ² and washed twice with 1 mL of warmed 0.5 mM EDTA/PBS(-). 2 mL of warmed 0.5 mM EDTA/PBS(-) was added and incubated at 37°C, 5% CO ₂ for 5 minutes. After incubation, EDTA/PBS(-) was removed with an aspirator, and 1 mL of undifferentiated maintenance medium containing 10 μM Y-27632 was added. The cells were pipetted until dissociated into single cells, and 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 undifferentiated maintenance medium. Y-27632 was added to 10 μM to suppress apoptosis, and a required amount of iMatrix-silk (Funakoshi Co., Ltd.) was added to allow cells to adhere (0.5 μL/well for 24-well plates), and the cells were seeded in 24-well plates at 2 × 10 ⁴ cells/cm ² and cultured at 37°C under 5% CO ₂ for 1 day. The human iPS cells used were the 1383D2, 1231A3, and/or 1383D6 strains.

(1) The differentiation induction medium for human iPS cells to anterior primitive streak cells was completely replaced with a differentiation induction medium (500 μL/well) containing first stage differentiation inducers, and the cells were cultured at 37°C under 5% _CO2 for 1 day to obtain anterior primitive streak cells.

(2) The differentiation induction medium from anterior primitive streak cells to presomitic mesoderm cells was completely replaced with a differentiation induction medium (1 mL/well) containing differentiation induction factors for the second stage. After 2 days of culture at 37°C and 5% _CO2 , the medium was completely replaced and washed once with warmed PBS(-). 300 μL/well of warmed Accutase (Funakoshi Co., Ltd.) was added and incubated at 37°C and 5% _CO2 for 3 minutes. The cells were pipetted until dissociated 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 × ¹⁰⁵ cells/mL was prepared using Essential 6 medium. The second stage differentiation inducer and Y-27632 (10 μM) were added to achieve the respective concentrations, and 1 μL of iMatrix-silk was added per well, and the cells were seeded on a 24-well plate at 1.5× ¹⁰⁵ cells/ ^cm2 (500 μL/well). The cells were cultured at 37℃ under 5% _CO2 for 1 day to obtain presomitic mesoderm cells.

(3) The differentiation induction medium for presomitic mesoderm cells to late presomitic mesoderm cells was completely replaced with a differentiation induction medium (1 mL/well) containing stage 3 differentiation inducers, and the cells were cultured at 37°C under 5% _CO2 for 1 day to obtain late presomitic mesoderm cells.

(4) The differentiation induction medium for late presomitic mesoderm cells to posterior intermediate mesoderm was completely replaced with a differentiation induction medium (1 mL/well) containing stage 4 differentiation inducers, and the cells were cultured at 37°C under ₅ % CO2 for 3 days to obtain posterior intermediate mesoderm.

The cells obtained expressed both WT1 (green), a posterior intermediate mesoderm marker, and HOXD11 (red), which indicates that they can differentiate into metanephros (Figure 2). In both figures, the light-colored areas indicate the stained areas.

(5) Induction of differentiation of posterior intermediate mesoderm cells into nephron progenitor cells The medium was completely replaced and washed once with warmed PBS(-). 300 μL/well of warmed Accutase was added and incubated at 37°C under 5% _CO2 for 3 minutes. The cells were pipetted until dissociated 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 × ¹⁰⁵ cells/mL was prepared using Essential 6 medium. Stage 5 differentiation inducers were added to achieve each concentration, and 100 μL of the cell suspension was seeded into a V-bottom or U-bottom 96-well plate (non-adhesive) (cell density was 1 × ¹⁰⁴ cells/well). The cells were centrifuged (160 g, 2 minutes) to easily form cell clusters, and the cells were precipitated. The cells were cultured at 37°C under 5% _CO2 for 3 days to obtain cell clusters of nephron progenitor cells.

(6) Induction of differentiation of nephron progenitor cells into nephron organoids The cell mass was placed on a filter and cultured at the air-liquid interface for 7 days or more to obtain nephron organoids. Essential 6 supplemented with 10% FBS was used as the medium.

The obtained nephron organoids were immunostained with antibodies against PODOCALYXIN (glomerular podocyte marker; white), LTL (Lotus tetragonolobus lectin) (proximal tubule marker; red), and E-cadherin (distal tubule marker, CDH1; green), as well as nuclear staining (blue). The images obtained are shown in Figure 3. Numerous dot-shaped signals positive for PODOCALYXIN (white) were observed within the organoids, and numerous LTL signals (pale in the black and white image) were observed adjacent to these signals. Furthermore, numerous E-cadherin signals (pale in the black and white image) were observed not only in the same areas as the LTL signals, but also in the center of the organoids.

These results revealed that a series of nephron structures was formed from the surface to the center of the organoid, consisting of a group of PODOCALYXIN-positive cells (glomerular podocytes), LTL-positive structures (proximal tubules), and E-CADHERIN-positive structures (distal tubules). This demonstrates that the posterior intermediate mesoderm produced in this example has the ability to differentiate into nephrons.

[Example 2] Examination of the differentiation induction period of the first stage It is known that when pluripotent stem cells are sequentially cultured in the presence of the factors listed in Figure 4, they differentiate predominantly into anterior intermediate mesoderm and further into Wolffian ducts (Costantini F. & Kopan R., Dev Cell. 18(5). 2010). Normally, nephron progenitor cells do not arise from anterior intermediate mesoderm, so if the proportion of cells heading toward anterior intermediate mesoderm (from pluripotent stem cells) can be reduced, the proportion of cells that will become posterior intermediate mesoderm will relatively increase, and as a result, it is expected that more nephron progenitor cells or nephron organoids can be obtained. Therefore, the culture period of the first stage was changed under the culture conditions in Figure 4 to examine conditions under which the proportion of cells heading toward anterior intermediate mesoderm from human iPS cells is reduced. After the end of the third stage, the expression of marker proteins of the Wolffian duct (GATA) and the tip of the Wolffian duct (RET) was analyzed by immunostaining.

As a result, as shown in Figure 5, when the first stage was cultured for 16 hours, the number of GATA3-positive cells was extremely high (left), but when it was cultured for 24 hours, the number of GATA3-positive cells decreased dramatically (right). This shows that a long culture period in the first stage leads to a decrease in the number of cells differentiating into anterior intermediate mesoderm.

Based on these results, we investigated the first stage culture period that would allow more pluripotent stem cells to be induced to differentiate into posterior intermediate mesoderm. Human iPS cells were cultured under the conditions described in Figure 6, and immunostained with anti-WT-1 antibody after the end of the fourth stage. The results showed that the number of WT-1 positive cells was significantly increased when the culture period was approximately 10 to 12 hours longer than the culture period (approximately 16 hours) that preferentially induced differentiation into anterior intermediate mesoderm (Figure 7). Furthermore, an investigation of multiple human iPS cell lines showed that the first stage culture periods that resulted in the highest proportion of cells that became posterior intermediate mesoderm were approximately 27 hours and approximately 32 hours for the 1383D2 and 1231A3 strains, respectively.

Therefore, it was found that by setting the first stage culture period to 17 to 40 hours, more preferably 20 to 38 hours, and even more preferably 24 to 36 hours, more human pluripotent stem cells can be induced to differentiate into posterior intermediate mesoderm.

[Example 3] Examination of conditions for inducing differentiation from nephron precursor cells to nephron organoids For the differentiation process from nephron precursors to nephron organoids, a more convenient culture method (not only air-liquid interface culture) was attempted. Human iPS cells were cultured under the conditions described in Figure 8, and the presence or absence of nephron organoids was observed 10 days after the start of culture. Specifically, the cells were cultured under the same conditions as in [Example 1] up to step (5), and in step (6), the nephron precursor cell mass was subjected to suspension culture (shaking at 50 rpm using a shaker on a low-adhesion 35 mm dish) in Essential 6 containing 0.5% polyvinyl alcohol (PVA) and 10% KSR. Two types of PVA with different molecular weights (MW: 30,000-70,000 and 85,000-124,000) were used.

As shown in Figure 9, regardless of the PVA used, a large number of nephron organoids of roughly uniform size were obtained. When a PVA with a high molecular weight (MW: 85,000-124,000, image on the right in Figure 9) was used, the PVA fibers tended to become thread-like and entangle the organoids, so a PVA with a lower molecular weight (for example, MW: 30,000-70,000, image on the left in Figure 9) appears to be preferable.

Therefore, it was demonstrated that the nephron progenitor cells obtained by the method of the present invention can be induced to differentiate into nephron organoids even by suspension culture. Furthermore, it was also demonstrated that the use of a PVA-containing medium in this process suppresses excessive aggregation, resulting in the production of multiple nephron organoids that are roughly the same size.

[Example 4] (4) Investigation of conditions for inducing differentiation of late pre-somitic mesoderm cells into posterior intermediate mesoderm Human iPS cells were cultured under the conditions shown in Figure 10 to induce differentiation into nephron organoids. These conditions were the same as those in the culture conditions (Figure 1) of [Example 1] in the medium used in step (4) except that the retinoic acid receptor agonist was omitted.
As shown in FIG. 11, nephron organoids were obtained even when cultured under the conditions in FIG. 10.
Therefore, (4) it was shown that retinoic acid receptor agonists are not essential for inducing differentiation of late presomitic mesoderm cells into posterior intermediate mesoderm, and that differentiation is possible with the presence of fibroblast growth factor and an activator of activin receptor kinase 4 and 7.

Claims (16)

(3) A method for producing late presomitic mesoderm cells, comprising the step of culturing presomitic mesoderm cells in a medium containing a fibroblast growth factor, an activator of activin receptor kinase 4, 7, a BMP inhibitor, and a GSK3β inhibitor. The method according to claim 1, wherein the fibroblast growth factor is FGF8, the activator of activin receptor kinase 4, 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 presomitic mesoderm cells are cells produced from pluripotent stem cells by a method comprising the following steps (1) and (2):
(1) culturing pluripotent stem cells in a medium containing an activator of activin receptor kinase 4, 7, and a GSK3β inhibitor to obtain an anterior primitive streak cell culture; and (2) culturing the anterior primitive streak cell culture in a medium containing a fibroblast growth factor, a TGFβ inhibitor, a BMP inhibitor, and a GSK3β inhibitor. The method according to claim 3, wherein in steps (1) and (2), the activator of activin receptor kinase 4, 7 is activin A, the GSK3β inhibitor is CHIR99021, the fibroblast growth factor is FGF8, the TGFβ inhibitor is A83-01, and the BMP inhibitor is LDN193189. The method according to claim 3 or 4, wherein the concentration of the GSK3β inhibitor in steps (1) to (3) is 1 to 5 μM. The method according to any one of claims 3 to 5, wherein the pluripotent stem cells are iPS cells. The method of claim 6, wherein the iPS cells are human iPS cells. A method for producing posterior intermediate mesoderm cells, comprising obtaining late presomitic mesoderm cells by the method according to any one of claims 1 to 7, and (4) culturing the late presomitic mesoderm cells in a medium containing fibroblast growth factor and an activator of activin receptor kinase 4 and 7. The method according to claim 8 , wherein in step (4), the fibroblast growth factor is FGF8 and the activator of activin receptor kinase 4, 7 is activin A. The method according to claim 8 or 9, wherein in step (4), the medium further contains a retinoic acid receptor agonist. The method according to claim 10 , wherein in step (4), the retinoic acid receptor agonist is TTNPB. A method for producing nephron progenitor cells, comprising: obtaining posterior intermediate mesoderm cells by the method according to any one of claims 8 to 11 ; and (5) culturing the posterior intermediate mesoderm cells in a medium containing a fibroblast growth factor, a GSK3β inhibitor, a BMP inhibitor, and a ROCK inhibitor. The method according to claim 12 , wherein in 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, comprising obtaining nephron progenitor cells by the method according to claim 12 or 13 , and further comprising (6) a step of culturing the nephron progenitor cells at an air-liquid interface. A method for producing a nephron organoid, comprising obtaining nephron progenitor cells by the method according to claim 12 or 13 , and further comprising (6') a step of suspension-culturing the nephron progenitor cells. The method according to claim 15 , wherein the step (6') is a step of carrying out suspension culture in a medium containing polyvinyl alcohol.

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