JP7421049B2 - Method for inducing glomerular podocytes and method for producing podocytes from pluripotent stem cells using the inducing method - Google Patents

Description

The present invention relates to a method for selectively inducing glomerular podocytes from nephron precursor cells. The present invention also relates to a method for producing glomerular podocytes from pluripotent stem cells using the induction method.

Podocytes, which are present in the glomeruli of the kidneys, play an important role in blood filtration. Podocyte research is an important research theme from a medical economics point of view, as damage to podocytes results in proteinuria, which eventually progresses to end-stage renal failure and requires dialysis treatment.

In podocyte research, immortalized human podocyte cell lines have been used for in vitro analysis (Non-Patent Document 1). However, this cell line has a problem in that the expression level of genes and proteins characteristic of podocytes is extremely low, making it an insufficient material for carrying out research (Non-Patent Document 2).
In addition, there have been reports in the past that podocytes were selectively induced from human iPS cells, but the expression level of NPHS1, which is supposed to be a podocyte-specific gene, was 1/5 to 1/2 that of human iPS cells before induction. The expression remains at an extremely low level of about 10 (Non-patent Documents 3 to 5; Patent Document 1). Therefore, the cells induced in these reports are considered to be of insufficient quality to be called podocytes.

In recent years, including the report by the present inventors, several studies have been reported in which nephron progenitor cells are induced from human iPS cells and renal organoids containing podocytes and renal tubular cells are created in vitro (Non-patent Document 6). ~9; Patent Document 2). Kidney organoids are a population containing many cell types, but cell-autonomous cell-based organoids using more purified podocytes are needed for podocyte injury model experiments and drug screening for the development of therapeutic agents to reduce proteinuria. It is necessary to verify the (cell-autonomous) effects. Therefore, there is a need for a method for efficiently inducing differentiation or producing a cell population consisting of highly pure podocytes.

US Patent Application Publication 2016/0143949 A1 WO2015/056756

M.A. Saleem et al., J. Am. Soc. Nephrol. 13, 630-638, 2002) S. Chittiprol et al., Am J Physiol Renal Physiol 301, F660-671, 2011 B. Song et al., PLoS ONE 7, e46453, 2012 O. Ciampi, et al., Stem Cell Res. 17, 130-139, 2016 S. Musah et al., Nat. Biomed. Eng. 1, 0069, 2017 Taguchi et al., Cell Stem Cell 14, 53-67, 2014 Takasato et al., Nature 526, 564-568, 2015 Morizane et al., Nat. Biotechnol. 33, 1193-1200, 2015 Taguchi et al., Cell Stem Cell 21, 730-746, 2017

The present invention aims to provide a method for inducing glomerular podocytes. Another object of the present invention is to provide a method for producing podocytes that can obtain a cell population with a high composition ratio of podocytes by selectively inducing podocytes.

As a result of extensive research to solve the above problems, the present inventors have determined that the differentiation process from nephron progenitor cells to podocytes is characterized by (1) the mesenchymal-to-epithelical transition (MET) stage (MET); Hereinafter, the renal epithelial precursor aggregate (pre-tubular aggregate: PA) formation stage), (2) Renal vesicle (RV) formation stage, and (3) nephron fraction formation stage (hereinafter, the As a result of examining the signals necessary for inducing podocytes into three stages (sometimes referred to as the podocyte formation period), they found that stage-specific adjustment of specific signals is important, and completed the present invention. did.
The present invention includes the following aspects.
[1] A method for inducing differentiation of podocytes, comprising the following steps:
Step A: culturing nephron progenitor cells or a population thereof in a medium containing a Wnt agonist (preferably a Glycogen Synthase Kinase (GSK)-3β inhibitor, more preferably CHIR99021) and a ROCK inhibitor (preferably Y27632) The cells or cell populations obtained here are called "renal epithelial precursor aggregates (pre-tubular aggregates: PV)"),
Step B: A step of culturing the cells or cell population obtained in Step A in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) (the cells or cell population obtained here are referred to as " called the renal corpuscle (RV), and
Step C: A step of culturing the cells or cell population obtained in Step B in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) (the cells or cell population obtained here are podocyte.),
Here, at least one of the media in step B or step C contains a TGFβ signal pathway inhibitor (preferably SB431542).
[2] In [1] above, the concentration of the Wnt agonist in the medium used in step A is more than 1 μM and less than 10 μM (preferably about 2 μM to about 8 μM, more preferably about 3 μM to about 5 μM). Method described.
[3] [1] above, wherein the medium in steps B and C further contains a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021) at a concentration of about 1 μM or less (preferably about 0.5 μM) Or the method described in [2].
[4] The culturing in step A is carried out for 12 to 48 hours (preferably 18 to 30 hours, more preferably 21 to 27 hours, still more preferably about 24 hours), and the culturing in step B is carried out for 12 to 48 hours. The method according to any one of [1] to [3] above, wherein the time is 48 hours (preferably 18 hours to 30 hours, more preferably 21 to 27 hours, still more preferably about 24 hours).
[5] The method according to [4] above, wherein the culturing in step C is carried out for 2 to 30 days (preferably about 3 to about 15 days, more preferably 4 to 10 days).
[6] The method according to any one of [1] to [5] above, wherein the Wnt agonist is CHIR99021 and the TGFβ signal pathway inhibitor is SB431542.
[7] The method according to any one of [1] to [6] above, wherein the nephron progenitor cells are nephron progenitor cells derived from mammalian-derived iPS cells.
[8] The method according to [7] above, wherein the iPS cells are human-derived iPS cells.
[9] The method according to [7] or [8] above, wherein the medium in step B and/or step C further contains a Wnt signal inhibitor (preferably IWR-1).
[10] The method according to any one of [7] to [9] above, wherein the medium in Step B and/or Step C further contains retinoic acid (RA) or an RA analog.
[11] The nephron progenitor cells undergo the following steps:
(a) culturing embryoid bodies derived from human iPS cells in a medium containing a high concentration (concentration A) of a Wnt agonist (preferably CHIR99021);
(b) culturing the embryoid bodies in a medium containing Bmp (preferably Bmp2, Bmp4 or Bmp7, more preferably Bmp4), activin, retinoic acid, and a medium concentration (concentration B) of a Wnt agonist; and (c) culturing the embryoid body in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) and a low concentration (concentration C) of a Wnt agonist;
in that order (wherein the concentration of the Wnt agonist is concentration A>concentration B>concentration C, where concentration A is at least 5 times greater than concentration C),
The method according to any one of [7] to [10] above, which is induced from human iPS cells by.
[12] The Wnt agonist in steps (a), (b) and (c) is CHIR99021, and the concentration A and the concentration C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively. , and the Bmp in step (b) is Bmp4 and the concentration in the medium is 1 ng/mL to 10 ng/mL, and the concentration of activin in the medium in step (b) is 2.5 ng/mL. The method according to [11] above, wherein the concentration is ~40 ng/mL.

[13] A medium kit for inducing differentiation of podocytes, the kit comprising a differentiation-inducing medium and three containers containing the respective components described below;
(1) a first container containing a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021) and a ROCK inhibitor (preferably Y27632);
(2) TGFβ signal pathway inhibitor (preferably SB431542), Wnt signal inhibitor (preferably IWR-1), retinoic acid (RA) or RA analog, and Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably (3) a TGFβ signal pathway inhibitor (preferably SB431542), a Wnt signal inhibitor (preferably IWR-1), retinoic acid (RA) or an RA analog, and Fgf9); (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9).
[14] The culture medium kit according to [13] above, wherein the containers (2) and (3) further contain a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021).

[15] A method for producing a podocyte cell population, comprising the following steps:
(i) inducing differentiation of nephron progenitor cells or populations thereof from mammalian-derived iPS cells (preferably human iPS cells) or populations thereof;
(ii) Nephron progenitor cells or a population thereof obtained in step (i) are treated in a medium containing a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021) and a ROCK inhibitor (preferably Y27632). a step of culturing (the cells or cell populations obtained here are referred to as "pre-tubular aggregates (PA)");
(iii) A step of culturing the cells or cell population obtained in step (ii) in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) (the cells or cell population obtained here). are called "renal vesicles (RV)"), and
(iv) A step of culturing the cells or cell population obtained in step (iii) in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) (the cells or cell population obtained here). is referred to as a "podocyte").
Here, at least one of the medium in step (iii) or step (iv) contains a TGFβ signal pathway inhibitor (preferably SB431542), and at least one of the medium in step (iii) or step (iv) contains a Wnt signal pathway inhibitor. contains an inhibitor (preferably IWR-1), and at least one of the media in step (iii) or step (iv) contains retinoic acid (RA) or an RA analog.
[16] The concentration of the Wnt agonist in the medium used in step (ii) is more than 1 μM and less than 10 μM (preferably about 2 μM to about 8 μM, more preferably about 3 μM to about 5 μM), [ 15].
[17] The medium in steps (iii) and (iv) further contains a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021) at a concentration of about 1 μM or less (preferably about 0.5 μM). The method described in [15] or [16] above.
[18] The method according to any one of [15] to [17] above, wherein the iPS cells are human-derived iPS cells.
[19] The culture in step (ii) is for 12 to 48 hours (preferably 18 to 30 hours, more preferably 21 to 27 hours, still more preferably about 24 hours), and the step (ii) According to any one of [15] to [18] above, wherein the culture is for 12 to 48 hours (preferably 18 to 30 hours, more preferably 21 to 27 hours, still more preferably about 24 hours). the method of.
[20] The method according to [17] above, wherein the culturing in step (iv) is carried out for 2 to 30 days (preferably about 3 to about 15 days, more preferably 4 to 9 days).
[21] The Wnt agonist is CHIR99021, the TGFβ signal pathway inhibitor is SB431542, and the Wnt signal inhibitor is IWR-1, according to any one of [15] to [18] above. Method.
[22] The step (i) is the following step:
(a) culturing embryoid bodies derived from human iPS cells in a medium containing a high concentration (concentration A) of a Wnt agonist (preferably CHIR99021);
(b) culturing the embryoid bodies in a medium containing Bmp (preferably Bmp2, Bmp4 or Bmp7, more preferably Bmp4), activin, retinoic acid, and a medium concentration (concentration B) of a Wnt agonist; and (c) culturing the embryoid body in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) and a low concentration (concentration C) of a Wnt agonist;
in that order (wherein the concentration of the Wnt agonist is concentration A>concentration B>concentration C, where concentration A is at least 5 times greater than concentration C),
The method according to any one of [15] to [21] above.
[23] The Wnt agonist in steps (a), (b) and (c) is CHIR99021, and the concentration A and the concentration C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively. , and the Bmp in step (b) is Bmp4 and the concentration in the medium is 1 ng/mL to 10 ng/mL, and the concentration of activin in the medium in step (b) is 2.5 ng/mL. The method according to [20] above, wherein the concentration is ~40 ng/mL.

[24] A method for evaluating the influence or effect of a test substance on podocytes, in the presence of the test substance, as described in any one of [1] to [12], [15] to [23] above. A cell population of podocytes prepared by the above method was cultured, and the following items regarding the cells were examined: cell proliferation, cell survival, cell disorder, protein expression level, gene expression level, albumin uptake ability, albumin filtration ability, autophagy. A method comprising measuring at least one item selected from the group consisting of activity and intracellular transmission signals and evaluating the effect of the test substance on podocytes.
[25] A method for screening for a substance that treats or recovers from podocyte disorder, wherein (x) is produced by the method described in any one of [1] to [12], [15] to [23] above. A step of culturing a podocyte cell population in the presence of a drug and test substance that exhibits podocyte-toxicity, and (y) reducing podocyte cell survival, proliferation or damage, changes in protein expression levels, and changes in gene expression levels. A method for screening candidate substances for treating or recovering podocyte disorders, the method comprising: measuring at least one item selected from the group consisting of;
Here, the culture of the podocyte cell population in step (x) may be any of the following:
(x-1) Cultivating a podocyte cell population in an environment in which a drug exhibiting podocytotoxicity and a test substance are simultaneously present, or (x-1) culturing a podocyte cell population in the presence of a drug exhibiting podocytotoxicity. , and then culturing the impaired podocyte cell population in the presence of a test substance.
[26] The screening method according to [25] above, wherein the drug exhibiting podocyte-toxicity is at least one drug selected from the group consisting of puromycin aminonucleoside, doxorubicin, angiotensin II, and anti-podocyte antibodies.

[27] A method for treating or preventing kidney and/or glomerular diseases, comprising:
(I) The podocyte cell population produced by the method described in any one of [1] to [12] and [15] to [23] above is applied to a part of the kidney tissue of a subject suffering from the disease. (II) in the transplanted cell population of podocytes, wherein the cell population of podocytes is optionally transplanted with stromal cells and/or vascular endothelial cells and/or a cell scaffold; migrating into and remaining in the glomerulus of at least a portion of the renal tissue, thereby regenerating the renal tissue and treating diseases of the kidney and/or glomerulus;
method including.
[28] The kidney and/or glomerular disease is a pathological condition accompanied by podocyte disorder, nephrotic syndrome, nephrosclerosis, diabetic nephropathy, chronic glomerulonephritis, a combination thereof, or a complication of these diseases. A certain method described in [27] above.
[29] The method according to [27] or [28] above, wherein the podocyte cell population is transplanted as part of human iPS cells or renal organoids induced to differentiate from the population.

According to the present invention, glomerular podocytes can be efficiently induced from nephron precursor cells.

FIG. 1 is a diagram illustrating a schematic example of a protocol for inducing podocytes of the present invention. The upper figure (a) shows an example of a method for inducing podocytes using mouse nephron progenitor cells, and the lower figure (b) shows an example of a method for inducing podocytes using nephron progenitor cells derived from human iPS cells. It shows. CHIR: CHIR99021; Y: Y27632; SB: SB431542; F: Fgf9; RA: Retinoic acid. FIG. 2 is a diagram showing the gene expression of Six2-GFP negative cells, Six2-GFP positive nephron progenitor cells (NPCs), and nephron progenitor cells (NPCs) selected with antibodies. FIG. 2 is a diagram showing an outline of an experiment to confirm the influence of a Wnt agonist (CHIR99021) using nephron progenitor cells (NPCs) prepared in Example 1. FIG. 2 is a diagram showing the results of treatment with a Wnt agonist (CHIR99021) at various concentrations and times in inducing podocytes using nephron progenitor cells (NPCs) prepared in Example 1. This is a micrograph taken on day 6. This is the result of confirming the presence of MafB-GFP-positive podocytes on day 6 when CHIR99021 was treated for 24 or 48 hours at a concentration of 3 μM or 5 μM in podocyte induction using nephron progenitor cells (NPCs). The upper figure shows the observation results using visible light and fluorescence. The figure below is a graph of the results. When nephron progenitor cells were treated with CHIR99021 for 24 hours, the time series (0 hours, These are the results confirmed after 24 hours and 48 hours). The results of confirming the gene expression of Six2, Lhx1, Pax8, and Cdh1 during the induction process are shown. These are the results of microscopic observation of the influence of growth factors or inhibitors on the process of inducing differentiation from renal epithelial precursor aggregates (PA) to renal corpuscles (RV). a: CHIR99021 (2 μM) and SB431542; b: IWR-1, Activin A, RA and BMS493. The upper row shows the results of observing cell growth using visible light, and the lower row shows the results of observing MafB-GFP-positive cells using fluorescence. These are the results of confirming the influence of growth factors or inhibitors on the process of inducing differentiation from renal epithelial precursor aggregates (PA) to renal corpuscles (RV). The percentage of MafB-GFP positive cells (a) and the number of podocytes (b) are shown. These are the results of examining gene expression by SB431542 treatment at the renal corpuscle (RV) stage (48 hours). These are the results of confirming the ratio and number of Wt1-positive proximal renal corpuscle (RV) cells after treatment with SB431542. We confirmed the influence of each factor (CHIR99021 (2 μM), IWR-1, activin, SB431542, retinoic acid (RA), and BMS493) on differentiation to podocyte lineage in STEP 3 after proximal renal corpuscle (RV) induction. These are the results.a: The upper row shows the results of observing cell growth using visible light, and the lower row shows the results of observing MafB-GFP-positive cells using fluorescence.b: The results of observing MafB-GFP-positive cells (podocytes) in the cell population. Show percentage. These are the results of confirming the effect of SB431542 on the cell number of each nephron (podocyte, PT, and DT) in STEP 3 after proximal renal corpuscle (RV) induction. These are the results of confirming the expression of podocyte-specific proteins and other renal tubular markers in podocytes produced using the method of the present invention. FIG. 2 is a schematic diagram of an example of applying the three-step stepwise podocyte differentiation induction method established for mouse NPCs to human iPS cells (hiPS). FIG. 3 is a diagram showing the results of confirming the influence of a Wnt agonist (CHIR99021) on STEP1 in inducing podocytes using nephron progenitor cells (NPCs) derived from human iPS cells (hiPS). FIG. 2 is a diagram showing the results of confirming the influence of addition of various factors in STEP 2 in inducing podocytes using nephron progenitor cells (NPCs) derived from human iPS cells (hiPS). FIG. 3 is a diagram showing the results of confirming the influence of addition of various factors in STEP 3 in inducing podocytes using nephron progenitor cells (NPCs) derived from human iPS cells (hiPS). These are the results of comparing podocytes induced using the method of the present invention and podocytes of kidney organoids. The figure on the left compares the proportions, and the figure on the right compares the number of podocytes. FIG. 2 is a diagram comparing the expression of podocyte-specific genes in induced podocytes, human adult kidney-derived podocytes, human iPS cells, and immortalized podocyte cell lines. FIG. 2 is a diagram showing the expression sites of WT1, NEPHERIN, NEPH1, and PODOCIN in induced podocytes. This is a photograph of induced podocytes observed with a transmission electron microscope. The arrow in the left image indicates the foot process present in the basolateral region. The arrow in the right figure indicates a slit membrane-like structure (filament-like bridge) that exists between podocytes. This is another photograph of induced podocytes observed with a transmission electron microscope. The arrow indicates the NEPHRIN molecule. These are the results of western blotting analysis of protein expression in induced podocytes. Shows the survival rate when induced podocytes were treated with puromycin aminonucleoside (PAN). These are the results of western blotting analysis of protein expression when induced podocytes were treated with puromycin aminonucleoside (PAN).

The invention will now be described by way of example embodiments, along with preferred methods and materials that can be used in the practice of the invention, but the invention is not limited to the embodiments described below. It should be noted that, unless otherwise specified herein, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Also, any materials and methods equivalent or similar to those described herein can also be used in the practice of the invention. Additionally, all publications and patents cited herein in connection with the invention described herein are incorporated herein by reference, e.g., as indicating methods, materials, etc. that may be used in the invention. It constitutes a part of the

In this specification, when the expression "X to Y" is used, it is used to mean that the lower limit is X and the upper limit is Y, or the upper limit is X and the lower limit is Y. In this specification, "and/or" is used to include either one or both. Further, in this specification, "about" is used to mean a tolerance of ±10%.

The kidney develops through a tripartite interaction of nephron progenitor cells (NPCs), the precursors of glomerular podocytes and renal tubules, ureteric buds, the precursors of collecting ducts and ureters, and interstitial cells. Nephron progenitor cells undergo mesenchymal-epithelial transition (MET) and develop into epithelial nephron structures, including podocytes, proximal tubules (PT), and distal tubules (DT). Nephron progenitor cells form renal progenitor aggregates (PAs) and then differentiate into renal corpuscles (RVs) with proximal-distal polarization by Wnt signals from the ureteric bud. The renal corpuscle (RV) extends along the proximal-distal axis, undergoes an S-shaped body, and differentiates into the nephron epithelium. Podocyte precursor cells are present in the WT1-positive proximal domain of the renal corpuscle (RV) and the Wt1-positive proximal end domain of the S-shaped body, and ultimately differentiate into MafB-positive/Nephrin-positive mature podocytes. .
Although it has been suggested that Wnt and Notch signals play some role in the formation process of the proximal-distal axis of the nephron, it is unclear what kind of signals are involved in the formation of nephron compartments, especially in the differentiation of podocytes. Met.

The present inventors found that the induction method in previous reports regarding the induction of podocytes does not go through nephron progenitor cells that have the ability to differentiate into all nephron fractions, and that We focused on the fact that a step-by-step study had not been conducted. More importantly, it has not been verified how close the cells produced by these induction methods are to real podocytes, and the validity and validity of the induction methods cannot be guaranteed. It happened to me.

Therefore, based on our previous reports, the present inventors induced nephron progenitor cells from human iPS cells that have the ability to differentiate into the whole nephron fraction (Non-Patent Document 6). The differentiation process from progenitor cells to podocytes is divided into three stages: (1) mesenchymal epithelial transition induction (MET) stage, (2) Renal vesicle (RV) formation stage, and (3) nephron fraction formation stage. We investigated the signals required for induction into podocytes and revealed that the phase-specific regulation of CHIR99021, SB431542, IWR-1, retinoic acid, and Fgf9 is important. Podocytes induced according to the present invention express NPHS1 (Nephrin) 10 ⁴ times higher than human iPS cells and 10 ⁵ times higher than immortalized cell lines, and other podocyte-related proteins are also expressed in iPS cells and immortalized cell lines. It showed an extremely high expression level compared to the cell line. Furthermore, the inventors isolated podocytes from human adult kidneys and compared induced podocytes with real podocytes. They found that induced podocytes contain podocyte-related proteins such as NPHS1 and NPHS2 (Podocin), which are similar to those of in vivo podocytes. It was confirmed that they were expressed at similar levels. That is, the induced podocytes according to the present invention exhibit approximately the same quality at the gene expression level as real podocytes in vivo, which proves the validity of the induction method of the present invention.

One embodiment of the present invention is a method for inducing podocytes from nephron progenitor cells, which includes three steps: induction of nephron progenitor cells into renal epithelial progenitor aggregates (PA), and PA to renal corpuscles (RVs). ) and from RV to podocytes.
Another embodiment of the present invention is a method for selectively inducing glomerular podocytes from nephron precursor cells using the above three steps.
Another embodiment of the present invention is a method for producing glomerular podocytes from nephron precursor cells using the above three steps.
Another embodiment of the present invention is a method for producing podocytes from mammalian-derived iPS cells (preferably human iPS cells), which comprises the steps of inducing nephron progenitor cells from the iPS cells; This is a method for producing podocytes that combines two steps of inducing podocytes from nephron precursor cells.
Further, one embodiment of the present invention is a culture medium kit for inducing differentiation of podocytes from nephron precursor cells, which includes containers containing various substances used in the above three steps, and a culture medium.
Another aspect of the present invention is to provide an experimental model of podocytes using podocytes induced by any of the methods described above.
Another embodiment of the present invention is a method for evaluating a substance that is cytotoxic to podocytes, using podocytes induced by any of the methods described above.
Another embodiment of the present invention is a method for screening for substances that improve or treat podocyte disorders or diseases related thereto, using podocytes induced by any of the methods described above.
Yet another embodiment of the present invention provides a method for treating kidney and/or glomeruli, comprising transplanting podocytes induced by any of the methods described above into a subject suffering from kidney and/or glomerular disease. This is a method for treating or preventing diseases.

The details of the present invention will be explained below.
FIG. 1 outlines the protocol of the present invention for deriving glomerular podocytes from nephron progenitor cells (NPCs). The upper figure shows induction from mouse-derived NPCs, and the lower figure shows induction from human-derived NPCs. In either case, podocytes are induced from NPCs through a three-step process.

Nephron progenitor cells The nephron progenitor cells used in the method of the present invention can be either prepared from a living body or derived from pluripotent stem cells, but are preferably derived from pluripotent stem cells. Particularly preferred are nephron progenitor cells derived from iPS cells, which are pluripotent stem cells. Further, the nephron progenitor cells used in the present invention may be those obtained by culturing nephron progenitor cells derived from iPS cells while maintaining their characteristics under specific conditions, or those which are frozen and preserved. Nephron progenitor cells can be induced from pluripotent stem cells with reference to known methods. For example, it can be prepared with reference to reports by the present inventors (Non-Patent Document 6; Patent Document 2). These documents are incorporated herein by reference.
Examples include, but are not limited to, the following steps.
(a) culturing embryoid bodies derived from pluripotent stem cells in a medium containing a high concentration (concentration A) of a Wnt agonist (preferably CHIR99021);
(b) culturing the embryoid bodies in a medium containing Bmp (preferably Bmp2, Bmp4 or Bmp7, more preferably Bmp4), activin, retinoic acid, and a medium concentration (concentration B) of a Wnt agonist; and (c) culturing the embryoid body in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) and a low concentration (concentration C) of a Wnt agonist;
Nephron progenitor cells are produced by a differentiation induction step (wherein, the concentration of the Wnt agonist is concentration A>concentration B>concentration C, and concentration A is at least 5 times the concentration C). can.

.
The embryoid bodies used in the above step (a) can be prepared by culturing pluripotent stem cells (eg, ES cells and iPS cells) in any medium, preferably a serum-free medium. For example, when using human pluripotent stem cells, it is preferable to use human iPS cells treated with Bmp4 and Rock inhibitor (Y27632) (further adding Fgf as necessary), but not limited thereto. Embryoid bodies are prepared, and more preferably, embryoid bodies are then treated with activin and Fgf. The concentration of Bmp4 in the medium can be, for example, 0.3 to 5 ng/mL, preferably 0.5 to 2 ng/mL, and the concentration of Rock inhibitor (Y27632) in the medium can be, for example, 1 to 5 ng/mL. The concentration of Fgf in the medium can be, for example, 0 to 20 ng/mL, and the concentration of activin in the medium can be, for example, 0 to 20 ng/mL. .1 to 5 ng/mL, preferably 0.5 to 1 ng/mL. Treatment with Bmp4 and Rock inhibitor (Y27632) and Fgf can be carried out, for example, for 1 to 2 days, preferably 1 day, and treatment with activin and Fgf can be carried out, for example, for 1 to 4 days, preferably 2 days. .

Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells and can be produced by introducing specific reprogramming factors in the form of DNA or proteins into somatic cells. , exhibit properties almost equivalent to ES cells (e.g., pluripotency and proliferation ability based on self-renewal) (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). The reprogramming factor is a gene specifically expressed in ES cells, its gene product or its non-coding RNA, a gene that plays an important role in maintaining the undifferentiated state of ES cells, its gene product or its non-coding RNA, or It may also be composed of low molecular weight compounds. Examples of genes included in reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-MYC, N-Myc, L-Myc, Nanog, Lin28, Fbx15, and ERas. , ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, and the like. These reprogramming factors may be used alone or in combination. In addition, when using the c-MYC gene as a reprogramming factor by introducing it into somatic cells, use an introduction method that has a low possibility that the introduced c-MYC gene will be integrated into the chromosome of the target cell after iPS cell creation. For example, but not limited to, introduction using a Sendai virus vector or an episomal vector is preferable.

In the above steps (a), (b), and (c), it is important that the concentrations A, B, and C of the Wnt agonist contained in the medium used in each step have a specific relationship. The concentration of Wnt agonist in the medium used in each step is such that Concentration A > Concentration B > Concentration C and Concentration A is at least 5 times Concentration C, but preferably Concentration A is at least 2 times Concentration B. and concentration B is at least twice concentration C, more preferably concentration A is at least three times concentration B, and concentration B is at least three times concentration C. The concentration C of the Wnt agonist in step (c) is not particularly limited as long as differentiation induction occurs, and is appropriately selected depending on the Wnt agonist used. For example, when CHIR99021 is used, it is preferably 0.1 to 3.0 μM. can be raised to 0.5 to 2.0 μM.
For example, when using CHIR99021, the combination of concentrations A, B, and C of the Wnt agonist in steps (a), (b), and (c), including but not limited to, concentration A is 6 to 20 μM, preferably 7 .5 to 15 μM, more preferably about 10 μM, concentration B is selected from 2 to 6 μM, preferably 2 to 4.5 μM, more preferably about 3 μM, and concentration C is 0.5 to 2 μM, preferably can be selected from 0.7 to 1.5 μM, more preferably about 1 μM.
The Wnt agonist used can be a Wnt agonist that can be used in the method of the invention described below.

As the Bmp used in step (b), any of the Bmps described above can be used, and the concentration of each Bmp compound used can be appropriately selected depending on the purpose of use. For example, when using Bmp4, Bmp4 of any origin can be used, but preferably human Bmp4 is used, and the concentration in the medium is not particularly limited as long as the effect of inducing differentiation can be obtained, but for example, 0.1 ~30 ng/mL, preferably 1~10 ng/mL. Furthermore, when using another Bmp compound, a concentration that can exhibit the same effect as that obtained when Bmp4 is used can be appropriately selected.

The activin used in step (b) may be of any origin, but preferably human activin A is used. Further, the concentration in the medium is not particularly limited as long as the effect of inducing differentiation can be obtained, but it can be, for example, 2.5 to 40 ng/mL, preferably 7.5 to 15 ng/mL.
The concentration of retinoic acid used in the step (b) in the medium is not particularly limited as long as the effect of inducing differentiation can be obtained; be able to.

As the Fgf used in step (c), any of the above-mentioned Fgf can be used, and the concentration of each Fgf compound used can be appropriately selected according to the purpose of use. For example, when using Fgf9, Fgf9 of any origin can be used, but human Fgf9 is preferred. The concentration in the medium is not particularly limited as long as the effect of inducing differentiation can be obtained, but it can be, for example, 1 to 25 ng/mL, preferably 2.5 to 10 ng/mL. Furthermore, when using another Fgf compound, a concentration that can exhibit the same effect as that obtained when Fgf9 is used can be appropriately selected.

A preferred combination in the above steps (a) to (c) is that the Wnt agonist is CHIR99021, the concentration A and the concentration C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively, and , the Bmp in step (b) is Bmp4 and the concentration in the medium is 1 ng/mL to 10 ng/mL, and the concentration of activin in the medium in step (b) is 2.5 to 40 ng/mL. This is a method for inducing nephron progenitor cells.

Induced pluripotent stem (iPS) cells may be derived from mammals, such as mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, cows, horses, goats, monkeys, or Examples include humans, preferably mice or humans.

Nephron progenitor cells prepared from a living body or derived from mammalian pluripotent stem cells can optionally be further subjected to a cell sorting step. Although not limited thereto, for example, sorting can be performed using a cell sorter using at least one marker selected from the group consisting of ITGA8, ROBO2, PDGFRa, PDGFRb, CXCR4, and NGFR, or any combination thereof.

Induction of Podocytes The induction of podocytes from nephron progenitor cells of the present invention consists of three steps A to C.
Step A: A step of culturing nephron progenitor cells or a population thereof in a medium containing a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021) and a ROCK inhibitor (preferably Y27632) (the cells obtained here or the cell population is referred to as "renal epithelial precursor aggregate (PV)"),
Step B: The cells or population thereof obtained in Step A are cultured in a medium containing a TGFβ signal pathway inhibitor (preferably SB431542) and Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9). step (the cells or cell population obtained here are called "renal vesicles (RV)"), and
Step C: The cells or population thereof obtained in Step B are cultured in a medium containing a TGFβ signal pathway inhibitor (preferably SB431542) and Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9). step (the cells or cell population obtained here are called "podocytes"),
However, if the medium in step B contains a TGFβ signal pathway inhibitor, the medium in step C may not contain a TGFβ signal pathway inhibitor, and if the medium in step C contains a TGFβ signal pathway inhibitor, then step The medium in B may be free of TGFβ signal pathway inhibitors.

Step A (STEP 1) : Induction of NPCs into renal epithelial precursor aggregates (PA) Step A is a step of culturing nephron precursor cells or a population thereof in a medium containing a Wnt agonist and a ROCK inhibitor (preferably Y27632). It is. The cells or cell populations obtained in this step are referred to herein as "renal epithelial precursor aggregates (PA)."
Wnt agonists are defined as agents that activate TCF/LEF-mediated transcription in cells. Wnt agonists are therefore selected from true Wnt agonists, inhibitors of intracellular β-catenin degradation and activators of TCF/LEF, which bind to and activate members of the Frizzled receptor family, including any and all of the Wnt family proteins. Ru. Wnt agonists also include Wnt signaling pathway inhibitors, GSK-3β inhibitors, Dkk1 antagonists, and the like.
A GSK-3β inhibitor is defined as a substance that inhibits the kinase activity (for example, the ability to phosphorylate β-catenin) of Glycogen Synthase Kinase (GSK)-3β protein, such as CHIR99021 (CAS number: 252917-06-9). ), BIO (CAS number: 667463-62-9), SB216763 (CAS number: 280744-09-4), R-Spondin1, and many others are already known.
The Wnt agonist used in the present invention is preferably CHIR99021, SB216763, BIO, R-Spondin1, A 1070722, Lithium carbonate, 3F8, SB 415286, TDZD 8, TWS 119, TCS 2002, Wnt3, W nt3a, Wnt9b, More preferably CHIR99021 or SB216763, still more preferably CHIR99021.

When using CHIR99021 as a Wnt agonist in step A, the concentration of CHIR99021 in the medium varies depending on the origin of the cells used, but is usually more than 1 μM and less than 10 μM, and the lower limit is preferably about 2 μM, more preferably The upper limit is preferably about 8 μM, more preferably about 6 μM, and even more preferably about 5 μM. The combination of upper and lower limits is preferably about 2 μM to about 8 μM, more preferably about 2 μM to about 6 μM, and even more preferably about 3 μM to about 5 μM. By setting the concentration of CHIR99021 in this range in Step A, NPCs can be efficiently differentiated into PA. In Step A, when using another Wnt agonist in place of CHIR99021, a concentration that exhibits an equivalent effect can be set using CHIR99021 as a reference.

The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK), but in the present invention, for example, Y27632, Fasudil hydrochloride, GSK 429286, GSK 269962, AS 1892802, H 1152 dihydrochloride. e, or HA 1100 hydrochloride can be used, preferably Y27632 or Fasudil hydrochloride, more preferably Y27632. When using Y27632, the concentration is 1 μM to 1000 μM, preferably 1 μM to 200 μM, more preferably 1 μM to 100 μM, and even more preferably about 10 μM. In Step A, when using another Rock inhibitor instead of Y27632, a concentration that shows equivalent effects can be set using Y27632 as a reference.

The culture time in step A varies depending on the origin of the cells used, but is usually about 12 hours to about 48 hours, preferably about 18 hours to about 30 hours, more preferably about 21 hours to about 27 hours, and even more preferably Approximately 24 hours. By setting the culture time in Step A within this range, NPCs can be efficiently differentiated into PA.

Step B: Induction of PA to renal corpuscles (RV) Step B is a step of culturing PA cells or populations thereof in a medium containing Fgf and optionally a TGFβ signal pathway inhibitor. The cells or cell populations obtained in this step are referred to herein as "renal vesicles."
The TGFβ signal pathway inhibitor included in at least one of Step B or Step C is defined as a substance that inhibits signal transmission from binding of TGFβ to a receptor to SMAD, for example, a TGFβ receptor. A large number of substances have been reported, including substances that inhibit binding to the ALK family and substances that inhibit phosphorylation of SMAD by the ALK family.
The TGFβ signal pathway inhibitor used in step B of the present invention is not particularly limited as long as the desired cell-inducing effect can be obtained, but examples include SB431542 (CAS number: 301836-41), which is an ALK inhibitor. -9) or A83-01 (CAS number: 909910-43-6), D4476, GW788388, LY364947, R268712, RepSox, SB505124, SB525334, or SD208, preferably SB431542 or A83-01, and SB431542 But More preferred.
When SB431542 is used as a TGFβ signal pathway inhibitor in Step B, the concentration of SB431542 in the medium in Step B varies depending on the origin of the cells used, but is usually 1 μM to 500 μM, preferably 2 μM to 100 μM, more preferably is 3 μM to 50 μM, particularly preferably about 25 μM when mouse-derived nephron progenitor cells are used in step A, and about 25 μM is particularly preferable when nephron progenitor cells derived from human pluripotent stem cells are used in step A. 5 μM is particularly preferred. In step B, by adding SB431542 at the above concentration, PA can be efficiently differentiated into RV having proximal polarity. In Step B, when using another component in place of SB431542, a concentration that exhibits an equivalent effect can be set with reference to SB431542.

Fibroblast growth factors (Fgf) include, for example, Fgf2, Fgf4, Fgf7, Fgf9, or Fgf20, which are produced with reference to methods known per se based on known amino acid sequence information. Alternatively, recombinant human Fgf protein purchased from R&D Systems etc. can also be used. The Fgf used is preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9 or Fgf20, and even more preferably Fgf9. The concentration of Fgf9 in the medium varies depending on culture conditions, etc., but is, for example, 1 ng to 500 ng/mL, preferably 2 ng to 200 ng/mL, more preferably 5 ng to 100 ng/mL, and still more preferably about 10 ng/mL. . When using another Fgf instead of Fgf9, a concentration that exhibits the same effect can be set using Fgf9 as a reference.

The medium of Step B may further include a Wnt agonist at a concentration of about 1 μM or less. Wnt agonists that can be used are similar to the Wnt agonists described in step A, but are preferably GSK-3β inhibitors, more preferably CHIR99021.
When using CHIR99021 as a Wnt agonist in Step B, the concentration of CHIR99021 in the medium varies depending on the origin of the cells used, but is about 1 μM or less, preferably about 0.7 μM or less, more preferably about 0.5 μM. When using another Wnt agonist instead of CHIR99021, a concentration that exhibits an equivalent effect can be set using CHIR99021 as a reference.

When using PA derived from human iPS cell-derived nephron progenitor cells in Step A, a Wnt signal inhibitor may be added to the medium in either Step B or Step C, thereby improving podocyte induction efficiency. However, it is more preferable to add a Wnt signal inhibitor to the culture medium in both Step B and Step C.
Examples of Wnt signal inhibitors include, but are not limited to, IWR-1, IWP-2, IWP-3, IWP-4, XAV939, FH535, C59, VS-507, and CCT031374, and are preferred. is IWR-1, IWP-2, or XAV939.

When IWR-1 is used as a Wnt signal inhibitor in Step B, the concentration of IWR-1 in the medium in Step B varies depending on the origin of the cells used, but is usually 0.1 μM to 50 μM, preferably 0. .2 μM to 10 μM, more preferably 0.5 μM to 5 μM, even more preferably about 1 μM to about 2 μM. When using another Wnt signal inhibitor instead of IWR-1, a concentration that exhibits an equivalent effect can be set using IWR-1 as a reference.

When using PA derived from human iPS cell-derived nephron progenitor cells in step A, it is also preferable to add retinoic acid (RA) or an RA analog to the medium in step B.
An example of the retinoic acid that can be used in Step B is all-trans retinoic acid (ATRA), which can be purchased from Sigma-Aldrich or the like. Furthermore, retinoic acid that is artificially modified while retaining the functions of natural retinoic acid may also be used.
When ATRA is used as retinoic acid in Step B, the concentration of ATRA in the medium varies depending on the culture conditions and is not particularly limited as long as the desired cells can be obtained, but is usually 1 nM to 100 μM, preferably 1 nM to 100 μM. , 10 nM to 50 μM, more preferably 100 nM to 20 μM, even more preferably about 1 μM to about 10 μM.

In step B, a retinoic acid analog can be used instead of retinoic acid. Examples of retinoic acid analogs include AGN193109, AM580, AM80, BMS453, BMS195614, AC 261066, AC55649, and Isotretinoin, with AGN193109 being particularly preferred. In Step B, when using an RA analog instead of RA, a concentration that exhibits an equivalent effect can be set using RA as a reference.

The culture time in step B varies depending on the origin of the cells used, but is usually about 12 hours to about 48 hours, preferably about 18 hours to about 30 hours, more preferably about 21 to about 27 hours, and even more preferably about It is 24 hours. By setting the culture time in Step B within this range, PA can be efficiently differentiated into RV.

Step C: Inducing RV to podocytes Step C is a step of culturing RV cells or a population thereof in a medium containing Fgf and optionally a TGFβ signal pathway inhibitor to induce podocytes.
The TGFβ signal pathway inhibitor that can be used in step C is similar to the TGFβ signal pathway inhibitor described in step B, preferably SB431542 or A83-01, more preferably SB431542.
The concentration of the TGFβ signal pathway inhibitor in the medium in Step C can be the same as that described in Step B.

The Fgf that can be used in Step C is the same as the Fgf described in Step B, but is preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9. Furthermore, the concentration of Fgf in the medium in step C can be the same as that described in step B.

The medium of Step C may further include a Wnt agonist at a concentration of about 1 μM or less. Wnt agonists that can be used are similar to the Wnt agonists described in step A, but are preferably GSK-3β inhibitors, more preferably CHIR99021.
When using CHIR99021 as a Wnt agonist in step C, the concentration of CHIR99021 in the medium varies depending on the origin of the cells used, but is about 1 μM or less, preferably about 0.7 μM or less, more preferably about 0.5 μM. When using another Wnt agonist instead of CHIR99021, a concentration that exhibits an equivalent effect can be set using CHIR99021 as a reference.

When podocytes are induced using PA induced from human iPS cell-derived nephron progenitor cells in Step A, a Wnt signal inhibitor may be added to the medium in either Step B or Step C, but Step B It is preferable to add a Wnt signal inhibitor to both the culture medium and step C.
The Wnt signal inhibitor used in Step C is similar to the Wnt signal inhibitor described in Step B, but is preferably IWR-1. Furthermore, when a Wnt signal inhibitor is added to the medium in step C, the concentration thereof is the same as that described in step B.

When using RV induced from human iPS cell-derived nephron progenitor cells in Step A and Step B, retinoic acid (RA) or an RA analog may be added to the medium in Step C. The RA or RA analog used in step C is the same as the RA or RA analog described in step B, and if RA or RA analog is added to the medium in step C, its concentration is as described in step B. It is similar to

The culture time in step C varies depending on the origin of the cells used, but is usually about 2 days to about 9 days, preferably about 3 days to about 8 days. The culture time in step C is usually about 2 days to about 10 days, preferably about 3 days to about 7 days, more preferably about 4 days when using mouse cells, and when using human cells. is usually about 5 days to about 30 days, preferably about 5 days to about 20 days, more preferably 6 days to 15 days, and even more preferably about 7 days to about 10 days. By keeping the culture time in Step C within this range, RV can be efficiently differentiated into podocytes.

As described above, when using human iPS cell-derived nephron progenitor cells, the present invention includes the following steps:
Step A: culturing nephron progenitor cells or a population thereof in a medium containing a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021) and a ROCK inhibitor (preferably Y27632);
Step B: The cells or population thereof obtained in Step A are treated with a TGFβ signal pathway inhibitor (preferably SB431542), a Wnt signal inhibitor (preferably IWR-1), RA or RA analog, and Fgf (preferably, A step of culturing in a medium containing Fgf2, Fgf9 or Fgf20, more preferably Fgf9), and
Step C: The cells or population thereof obtained in Step B are treated with a TGFβ signal pathway inhibitor (preferably SB431542), a Wnt signal inhibitor (preferably IWR-1), RA or RA analog, and Fgf (preferably, culturing in a medium containing Fgf2, Fgf9 or Fgf20, more preferably Fgf9);
however,
If the medium in step B contains a TGFβ signal pathway inhibitor, the medium in step C may not contain a TGFβ signal pathway inhibitor, and if the medium in step C contains a TGFβ signal pathway inhibitor, the medium in step B The medium may be free of TGFβ signal pathway inhibitors,
If the medium in Step B contains a Wnt signal inhibitor, the medium in Step C may not contain a Wnt signal inhibitor, and if the medium in Step C contains a Wnt signal inhibitor, the medium in Step B may contain Wnt signal inhibitors. Does not need to contain signal inhibitors,
If the medium in step B contains RA or RA analog, the medium in step C may not contain RA or RA analog.
It is also a method for inducing differentiation of podocytes.

The medium used in the present invention can be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include, but are not limited to, DMEM (Dulbecco's modified Eagle's medium), DMEM/F12 medium, GMEM (Glasgow MEM) medium, Ham's F12 medium, IMDM ( Iscove's modified Dulbecco's medium), αMEM (Eagle's minimal essential medium α modified version), and mixtures thereof.

The medium used in the present invention may contain serum or may be serum-free. The medium used in the present invention may contain, if necessary, for example, albumin, ITS (insulin, transferrin, selenium), N-2 supplement (Thermo Fisher Scientific), B-27 (registered trademark) supplement minus vitamin A (Thermo Fisher Scientific). ), 2-mercaptoethanol, 1-thioglycerol, amino acids, L-glutamine, non-essential amino acids, ascorbic acid, and antibiotics including penicillin-streptomycin.

Media Kit One embodiment of the present invention is a media kit for inducing podocytes from nephron progenitor cells. The medium kit consists of a differentiation induction medium and three mediums containing each of the following components (1) to (3).
(1) Wnt agonists and ROCK inhibitors, (2) TGFβ signal pathway inhibitors and Fgf, and (3) TGFβ signal pathway inhibitors and Fgf.
The above (2) may further include a Wnt signal inhibitor and retinoic acid (RA) or an RA analog, and the above (3) may further include a Wnt signal inhibitor and a retinoic acid (RA) or an RA analog. However, when the nephron progenitor cells are derived from human iPS, it is preferable to include these components.
The components (1) to (3) above are preferably stored in separate containers. Therefore, the culture medium kit that is one aspect of the present invention includes a kit consisting of a differentiation-inducing medium and three containers containing each of the components (1) to (3) above.

The Wnt agonist, ROCK inhibitor, TGFβ signal pathway inhibitor, Fgf, Wnt signal inhibitor, and retinoic acid (RA) or RA analog used in the culture medium kit of the present invention are those described in the method for inducing podocytes of the present invention. It has the same ingredients as the one above. The Wnt agonist is preferably a GSK-3β inhibitor, more preferably CHIR99021, the ROCK inhibitor is preferably Y27632, the TGFβ signal pathway inhibitor is preferably SB431542, and the Fgf is preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9, and the Wnt signal inhibitor is preferably IWR-1.
The above components, Wnt agonist, ROCK inhibitor, TGFβ signal pathway inhibitor, Fgf, Wnt signal inhibitor, and retinoic acid (RA) or RA analog, are at concentrations used in the podocyte induction method of the present invention. It can be used by adding it to the culture medium.

The above-mentioned components may be provided in a state in which only the components are stored in their respective containers, or in a state in which a liquid medium or a powdered medium containing those components is stored in their respective containers.
The above medium kit of the present invention may further be combined with a medium or a kit thereof for inducing nephron progenitor cells from pluripotent stem cells, and combinations of such kits are also part of the present invention.

Production of Podocytes One embodiment of the present invention is a method for producing a cell population of podocytes from mammalian-derived iPS cells, which includes the three-step induction process described above. Therefore, the present invention includes the following aspects.
(i) inducing differentiation of nephron progenitor cells or populations thereof from mammalian-derived iPS cells (preferably human iPS cells) or populations thereof;
(ii) culturing the nephron progenitor cells or a population thereof obtained in step (i) in a medium containing a Wnt agonist and a ROCK inhibitor;
(iii) culturing the cells or population thereof obtained in step (ii) in a medium containing a TGFβ signal pathway inhibitor, a Wnt signal inhibitor, Fgf, and optionally further retinoic acid (RA) or an RA analog; as well as,
(iv) culturing the cells or a population thereof obtained in step (iii) in a medium containing a TGFβ signal pathway inhibitor, a Wnt signal inhibitor, Fgf, and optionally further retinoic acid (RA) or an RA analog;
however,
When the medium in step (iii) contains a TGFβ signal pathway inhibitor, the medium in step C may not contain a TGFβ signal pathway inhibitor, and when the medium in step C contains a TGFβ signal pathway inhibitor, step The medium in B may be free of TGFβ signal pathway inhibitors,
When the medium in step (iii) contains a Wnt signal inhibitor, the medium in step (iv) may not contain a Wnt signal inhibitor, and when the medium in step (iv) contains a Wnt signal inhibitor, The medium in step (iii) may not contain a Wnt signal inhibitor,
If the medium in step (iii) contains RA or RA analog, the medium in step (iv) may not contain RA or RA analog.
A method for producing a cell population of podocytes from iPS cells, comprising:

The Wnt agonist, ROCK inhibitor, TGFβ signal pathway inhibitor, Wnt signal inhibitor, Fgf, and retinoic acid (RA) or RA analog used in the above production method are those described in the podocyte induction method of the present invention. The same is true. The Wnt agonist is preferably a GSK-3β inhibitor, more preferably CHIR99021, the ROCK inhibitor is preferably Y27632, the Fgf is preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9, and the TGFβ signal pathway The inhibitor is preferably SB431542 and the Wnt signal inhibitor is preferably IWR-1.
The concentration of each of the above components, Wnt agonist, ROCK inhibitor, TGFβ signal pathway inhibitor, Fgf, Wnt signal inhibitor, and retinoic acid (RA) or RA analog, in the medium, and the concentration of the medium containing these components. Regarding the culture period, the description in the method for inducing podocytes of the present invention can be applied as is.
Furthermore, the description regarding the induction of nephron progenitor cells from iPS cells described in connection with the podocyte induction method of the present invention can be applied to step (i) in the above production method as is.

The podocyte cell population produced using the podocyte production method of the present invention means a cell population consisting of cells that are substantially podocytes. About 40% or more, preferably about 50% or more, more preferably about 60% or more, even more preferably about 70% or more, even more preferably about 80% or more, and most preferably about 90% or more is composed of podocytes. Refers to a cell population.
Whether or not the cells in the cell population are podocytes and their proportion can be confirmed by a known method using a podocyte cell marker. Examples of cell markers include, but are not limited to, NPHS1, NPHS2, WT1, MAFB, or SYNPO. Detection of the constituent ratio can be measured using, for example, a cell sorter or immunostaining.

Podocytes produced using the podocyte production method of the present invention are characterized in that they have similar expression of NPHS1, NPHS2, WT1, MAFB, and SYNPO compared to authentic podocytes (podocytes derived from human adults). . "Similar expression with respect to the expression of NPHS1, NPHS2, WT1, MAFB and SYNPO" refers to the expression of at least two, preferably at least three, more preferably four, and even more preferably all of these five genes. and/or equivalent expression in terms of protein expression. Equivalent expression means about 40% or more, preferably about 50% or more, more preferably about 60% or more, even more preferably about 70% or more, compared to the expression of genes and/or proteins in human adult-derived podocytes. More preferably, it shows expression of about 80% or more, most preferably about 90% or more.

Evaluation or screening method using podocytes One embodiment of the present invention is a cell model for evaluation or screening using podocytes produced using the above-described podocyte induction method or production method. This is an evaluation or screening method using a cell model.
The evaluation method of the present invention is a method that includes culturing podocytes or a cell population thereof produced using the above-mentioned podocyte induction method or production method with a target test substance, and confirming the effect on the podocyte cells. be. Effects on podocyte cells include, but are not limited to, cell proliferation, cell survival, cell damage, protein expression level, gene expression level, albumin uptake ability, albumin filtration ability, autophagy activity, or intracellular communication. Evaluation can be performed by measuring at least one item selected from the group consisting of signals. This makes it possible to evaluate the toxicity of the target substance to podocyte cells. The test substance to be used is not particularly limited, and examples thereof include low-molecular compounds, middle-molecular compounds, high-molecular compounds, amino acids, peptides, proteins, and natural extracts.
The above evaluation items can be measured with appropriate reference to known methods. Reagents and kits for measuring these evaluation items are also commercially available, and can be used without restriction.
By using the evaluation method of the present invention, it is possible to evaluate, for example, but not limited to, the toxicity of a drug that may cause renal damage.

Examples of the screening method of the present invention include a method of screening for substances that treat or recover from podocyte disorders. Candidate substances can be screened by performing the following steps using podocytes or cell populations thereof produced using the podocyte induction method or production method described above.
(x) a step of culturing in the presence of a podocyte-toxic agent and a test substance; and (y) a step of measuring survival or proliferation of podocyte cells.
The podocyte cell population in step (x) may be cultured in an environment where (x-1) a drug exhibiting podocyte-toxicity and a test substance are present simultaneously; ) A podocyte cell population may be cultured in the presence of an agent exhibiting podocyte-toxicity, and then the impaired podocyte cell population may be cultured in the presence of a test substance. In the latter case, the test substance may be added after removing the podocyte-toxic agent, or the test substance may be added without removing it.
Examples of drugs exhibiting podocytotoxicity include, but are not limited to, puromycin aminonucleoside, doxorubicin, angiotensin II, anti-podocyte antibodies including anti-nephrin antibodies, and preferably puromycin aminonucleoside, Or doxorubicin.
Further, the test substance is not particularly limited, and examples thereof include low-molecular compounds, middle-molecular compounds, high-molecular compounds, amino acids, peptides, proteins, and natural extracts.
By using the screening method of the present invention, it is possible to search for, for example, but not limited to, a drug that protects podocytes.

Furthermore, using the podocyte induction method or production method of the present invention, a podocyte cell population can also be produced from iPS cells derived from a patient with a podocyte-related disease. By using the podocyte cell population produced in this way, it is possible to evaluate and screen drugs for treating diseases related to podocytes. Such evaluation or screening methods are also part of the present invention.

Treatment of Kidney and Glomerular Diseases One embodiment of the present invention is a method for treating or preventing kidney and/or glomerular diseases using podocytes produced using the podocyte induction method or production method described above. . Therefore, the present invention provides (I) transplantation of a podocyte cell population produced by the above-described podocyte induction method or production method into a part of kidney tissue of a subject suffering from a disease, and (II) transplanted podocyte cells. at least some of the cells in the population migrate into and remain in the glomerulus of at least a portion of the renal tissue, thereby regenerating the renal tissue and treating renal and/or glomerular diseases. It is also a method that includes.

The subject suffering from the disease is not particularly limited as long as it is a mammal, and examples include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, cows, horses, goats, monkeys, and humans. , preferably human.
In the transplantation of podocytes into a portion of kidney tissue in (I) above, the podocyte cell population can also be transplanted together with other cells or cell scaffolds that support podocyte transplantation. Examples of other cells include, but are not limited to, interstitial cells and vascular endothelial cells. As the cell scaffold, any known or commercially available scaffold can be used as appropriate, and for example, a scaffold made of a biocompatible substance (also referred to as biomaterial) can be used. Examples of biomaterials include, but are not limited to, silk fibroin, polyethylene oxide, polyethylene glycol, fibronectin, keratin, polyaspartic acid, polylysine, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid, and polystyrene. Mention may be made of hydroxyalkanoates, dextran, PHA, collagen, chitosan, alginates, and other biocompatible polymers.

Podocytes induced according to the invention can also be transplanted with or as part of renal organoids. Kidney organoids are kidney-like structures with a higher-order structure, including differentiated nephrons consisting of glomeruli and tubules, nephron progenitor cells, interstitial cells, and a dendritic branching structure of collecting ducts that connect the differentiated nephrons to each other. It is an organization.

The above kidney and glomerular diseases include, but are not limited to, pathological conditions accompanied by podocyte disorder, nephrotic syndrome, nephrosclerosis, diabetic nephropathy, chronic glomerulonephritis, or combinations thereof, or diseases thereof. Preferably, it is a pathological condition accompanied by podocyte disorder.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to the following Examples.
(Materials and methods)
mouse
MafB-GFP knock-in mice and Six2-GFP-Cre transgenic mice were provided by Dr. Satoshi Takahashi of the University of Tsukuba and Dr. Andrew McMahon of the University of Southern California. Mice were housed in plastic cages on a CE-2 radiation-sterilized diet with a 12-h light cycle. All analyzes were performed using a minimum of three littermates. For analysis of the embryonic stage, noon on the day a vaginal plug was confirmed in the mated female was considered to be 0.5 day of the embryonic stage (E). All animal experiments were performed according to protocols approved by the Kumamoto University School of Medicine Animal Experiment Committee.

Podocyte induction from mouse nephron progenitor cells (NPCs)
Metanephros were isolated from MafB-GFP embryos at E15.5, minced in PBS using forceps, and dissociated by incubation in 0.25% trypsin-EDTA for 8 minutes at 37°C. After that, the cells were subjected to primary antibody staining using anti-Robo2 antibody and anti-Pdgfrβ antibody on ice, and then after performing secondary antibody staining, the Robo2 ^-high /Pdgfrβ ^- /Mafb-GFP ^- population was sorted using FACS. did. For Six2-GFP-Cre embryos, NPCs were sorted as Six2-GFP ⁺ cells or Robo2 ^-high /Pdgfrβ ^- /Podocalyxin ^- cells. In a 96-well low cell adhesion U-bottom plate, 100,000 NPCs were seeded in differentiation medium (150 μL) supplemented with predetermined concentrations of CHIR99021 and Y27632 (10 μM). After the cells were centrifuged to stick to the bottom, they were cultured at 37°C in a 5% CO ₂ incubator. After initial CHIR99021 induction, aggregated cells were transferred onto transwell inserts filled with serum-free differentiation medium supplemented with Fgf9 (10 μg/mL) and cultured with the following growth factors or inhibitors: CHIR99021 (2 μM ), IWR-1 (2 μM), activin A (10 ng/mL), SB431542 (25 μM), retinoic acid (10 μM), BMS493 (10 μM). The medium was changed every 2 days, and on the 6th day, the induced tissues were analyzed. As the serum-free differentiation medium, DMEM/F12 medium supplemented with 1% (v/v) Insulin-Transferrin-Selenium and 1% (v/v) penicillin-streptomycin was used.

Cell Culture The 201B7 hiPS cell line from a healthy female donor was maintained on iMatrix-511 silk in StemFit AK02N medium. Cells were cultured at 37°C with 5% _CO2 and passaged every 6-7 days. Medium was changed every 2, 4, 5, and/or 6 days. The NPHS1-GFP knock-in hiPS cell line was generated according to a previous report (Shamin et al. J. Am. Soc. Nephrol. 27, 1778-1791, 2016) and maintained in the same manner. Human immortalized podocyte cell lines were purchased from Bristol University. Cells were grown in RPMI medium supplemented with 10% FBS at 33°C with 5% _CO2 and passaged for 4-5 days. Induction to the podocyte phenotype was performed by culturing cells at 37°C and 5% _CO2 for 14 days.

Induction of podocytes from nephron progenitor cells (NPCs) derived from human iPS cells (hiPSCs) Spheroids containing NPCs were induced from hiPSCs according to the report by Taguchi et al. (Non-Patent Document 6 and Non-Patent Document 9). The induced spheroids were dissociated into single cells using Accumax by incubating for 8 minutes at 37°C. The dissociated cells were subjected to primary antibody staining with anti-ITGA-8 antibody and anti-PDGFRa antibody for 30 minutes on ice, followed by secondary antibody staining, and then NPCs were sorted as ITG8 ⁺ /PDGFRa ⁻ population using FACS. In a 96-well low cell adhesion U-bottom plate, 50,000 to 100,000 NPCs were seeded in serum-free differentiation medium (150 μL) supplemented with CHIR99021 (3 μM) and Y27632 (10 μM). After the cells were centrifuged to stick to the bottom, they were cultured at 37°C in a 5% CO ₂ incubator. After 24 hours, aggregated cells were transferred onto transwell inserts filled with serum-free differentiation medium supplemented with Fgf9 (10 μg/mL), IWR-1 (2 μM), SB431542 (5 μM), and retinoic acid (10 μM). The cells were cultured in a medium supplemented with the STEP 2 factor (factor B in the above step). After 48 hours of culture, the differentiated cells were transferred to a medium supplemented with STEP 3 factors (factor C in step above) containing IWR-1 (2 μM) and SB431542 (5 μM). The medium was changed every 3 days. On days 9-12, the induced podocytes were collected and analyzed.

Flow Cytometry Analysis Tissues derived from mouse or human NPCs were dissociated by treatment with 0.25% trypsin-EDTA for 8 minutes. Blocking was performed using normal mouse serum, staining was performed in 1×HBSS buffer containing 1% BSA and 0.035% NaHCO ₃ for 30 minutes, and then staining was performed with secondary antibody for 10 minutes. The stained cells were analyzed using FACS. Information on the antibodies used is shown in the table below.

Collection of podocytes from human adults After obtaining informed consent and approval from the Ethics Committee of the Graduate School of Life Sciences, Kumamoto University, human adult podocytes were collected from normal sites of kidney tissue removed from three kidney cancer patients. Kidney tissue was obtained. The patients were a 49-year-old woman, a 69-year-old man, and an 80-year-old man. All patients had normal renal function and normal urinary findings. Normal sections of renal tissue removed from patients were immersed in high glucose DMEM containing 10% FBS on ice immediately after removal. Glomeruli were isolated and digested with an enzyme mixture (Collagenase XI: 1 mg/mL, Dispase: 2.4 U/mL), followed by 0.25% trypsin-EDTA. After the cell dissociation process, RBC lysis buffer was used to disrupt the red blood cells. Thereafter, primary antibody staining was performed with an anti-human Nephrin antibody and an anti-human Podocalyxin antibody, followed by secondary antibody staining, and then human normal podocytes were isolated as a Nephrin ⁺ /Podocalyxin ⁺ population using FACS.

Quantitative RT-PCR and RNA sequence analysis
Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen), and reverse transcription was performed using random primers and the Superscript VILO cDNA synthesis kit (Life Technology). Quantitative PCR was performed using the Dice Real Time System Thermal Cycler (Takara Bio) and Thunderbird SYBR qPCR Mix (Toyobo). The used primers (forward primer and reverse primer) correspond to the following genes. Mouse: b-Act, Six2, Pax2, Sall1, Cited1, Robo2, Lhx1, Pax8, Cdh1, WT1, MafB, Foxc2, Pou3f3, Dkk1, Sox9, Nphs1, Nphs2, Cldn1, Lrp2, Slc34a1, Cdh6; Human: B- ACT, NPHS1, NPHS2, WT1, SYNPO. All samples were normalized by β-actin expression.

Immunohistochemical analysis Samples were fixed in 10% formalin, embedded in paraffin, and 6 mm thick sections were obtained. Antigen retrieval was performed with citrate buffer at pH 6.0. Sections were blocked with PBS containing 1% BSA and incubated with primary antibodies overnight at 4°C. It was stained with a secondary antibody conjugated with Alexa488, 568, 594, 633 or 647. Nuclei were stained with DAPI. Fluorescence images were captured with a confocal microscope. Information on the antibodies used is shown below.

Electron microscopy imaging Observations were performed using an immunoelectron microscope. As the antibody, an antibody against the N-terminal domain of Nephrin was used. The prepared sections were incubated with the antibody overnight and then incubated with a secondary antibody conjugated with 12 nm gold particles. After immunostaining, the sections were fixed and imaged using a JEM1230 transmission electron microscope (JEOL).

(result)
Example 1: Purification of nephron progenitor cells from mouse embryonic kidney
To analyze the signals required for the differentiation of NPCs into podocytes, we established a culture system that can detect the differentiation of NPCs purified from mouse embryonic kidneys into nephron epithelium and podocytes. According to previous reports, NPCs express Robo2 and are negative for Pdgfrb. Therefore, we performed flow cytometry analysis using Six2-GFP transgenic mice and confirmed that the Robo2 ^-high /Pdgfrb ^- fraction in the kidney at E15.5 was Six2-GFP ⁺ NPCs. In addition, we established a method to isolate Six2-GFP+ NPCs with high purity by excluding the Podocalyxin ⁺ fraction to remove podocytes present in the Robo2 ^-high /Pdgfrb ^- fraction. To quantitatively evaluate podocyte induction, we used MafB-GFP knock-in mice (MafB-GFP mice) that express GFP only in differentiated podocytes. As expected, the Robo2 ^-high /Pdgfrb ^- /MafB-GFP ^- cell fraction expresses NPC-specific genes such as Six2, Pax2, Sall, and Cited1 at levels comparable to those of Six2-GFP ⁺ cells, making it possible for NPCs to It was confirmed that the product could be isolated with high purity. The results are shown in Figure 2.

Example 2: Examination of Wnt signals essential for MET and differentiation into podocytes It has been reported that Wnt signals are required for MET of NPCs in vivo and in vitro (Stark et al. Nature 372, 679-83 , 1994, Kispert et al. Development 125, 4225-4234, 1998, Carrol et al. Dev. Cell 9, 283-292, 2005, Kuure et al. J. Am. Soc. Nephrol. 18, 1130-9, 2007 ). However, it was unclear how the intensity and duration of Wnt signals affect differentiation into podocytes.
First, we investigated the concentration and duration of Wnt signals required to induce mesenchymal-epithelial transition from NPCs. The NPCs prepared in Example 1 were treated with CHIR99021 (CHIR) as a Wnt agonist at a concentration of 1 to 10 μM for 12 to 48 hours, and analyzed on the 6th day. An overview is shown in Figure 3. After initial CHIR treatment, 0.5 μM CHIR was continuously added as basal medium to maintain tissue growth and survival. We found that treatment of NPCs with a combination of 3-5 μM CHIR for 24-48 hours resulted in MET and epithelialized nephron tissue formation. The results of microscopic observation are shown in FIG. When Mafb-GFP+ cells were confirmed, as shown in Figure 5, CHIR treatment at 3 μM for 24 hours significantly induced Mafb-GFP+ podocytes, while treatment at 5 μM and 48 hours hardly induced podocytes. Ta. From this, it was found that when NPCs derived from mouse embryonic kidneys were used, conditions of treatment with 3 μM CHIR for 24 hours were optimal. These results suggest that there is an optimal concentration and duration of Wnt treatment during epithelialization of NPCs, and further indicate that early Wnt signals significantly influence final podocyte formation. ing.

Example 3: Inhibition of TGF-β signaling in differentiation of renal epithelial progenitor aggregates (PA) to renal corpuscles (RV)
In order to study the induction process from NPCs to podocytes, the present inventors defined an intermediate step in the above in vitro culture system. The results of qRT-PCR are shown in Figure 6. This revealed that 24 hours after the start of induction corresponded to the PA differentiation stage, and 48 hours corresponded to the RV differentiation stage.

Since polarization has already occurred in the RV along the proximal-distal axis and the proximal region of the RV is presumed to contain podocyte precursors, direct differentiation of NPCs into the proximal RV may result in podocytes. The following study was conducted based on the hypothesis that the induction efficiency would be increased.
The effects of growth factors or inhibitors in the differentiation process from PA to RV (24 hours to 48 hours: STEP 2) were confirmed. A high concentration of CHIR (2 μM) inhibited the final podocyte percentage compared to the reference condition of 0.5 μM CHIR. In addition, IWR-1, a Wnt signal inhibitor, inhibited epithelialization. In contrast, SB431542 (SB), a Tgf-β signaling pathway inhibitor, significantly increased the percentage of final podocytes. Similar studies on other factors revealed that activin A, retinoic acid (RA), and the RA antagonist BMS493 did not increase the efficiency of podocyte induction. The results are shown in FIGS. 7 and 8.

When we examined the gene expression profile at the RV stage (48 hours), we found that SB treatment enhanced the expression of proximal region markers Wt1, MafB, and Foxc2 (Figure 9). On the other hand, the distal marker of RV was slightly decreased compared to the reference condition (0.5 μM CHIR). Furthermore, SB treatment increased both the proportion and number of Wt1 ⁺ proximal RV cells. On the other hand, high concentration CHIR treatment resulted in an increase in Wt1 ⁻ cells and a decrease in proximal RV cells. The results are shown in FIG.
These results suggest that inhibition of TGF-β signaling during the PA to RV process promotes proximal RV differentiation and, as a result, increases the proportion and number of terminal podocytes. .

Example 4: Other effects of inhibition of TGF-β signal after RV formation process Next, after differentiation into proximal RV, in the culture process from 48 hours to day 6 (STEP 3) during differentiation into each nephron site. We examined the effects of growth factors on podocyte induction. The results are shown in FIG. Continuous addition of SB from 48 hours to day 6 significantly increased MafB-GFP ⁺ podocytes on day 6. On the other hand, addition of 2 μM CHIR completely inhibited differentiation into podocytes. On the other hand, Wnt signal inhibitors did not significantly affect the proportion of podocytes. This result suggests that in mice, suppressing Wnt signals does not promote differentiation into podocytes, but strong Wnt signals suppress differentiation into podocytes. Other factors, activin and RA signal agonist/antagonist, had no significant effect on the proportion of podocytes compared to baseline conditions (0.5 μM CHIR).

Examination of cell counts on day 6 revealed that while SB treatment had no significant effect on the final number of podocytes, the number of LTL-positive proximal tubular cells decreased. The number of Cdh1-positive distal tubule cells tended to decrease, but the difference was not significant. The results are shown in FIG. Furthermore, gene expression analysis revealed that the expression of podocyte-related genes increased, while the expression of genes related to nephron fractions other than podocytes decreased. The results are shown in FIG.
Podocytes produced using the highly efficient induction method of the present invention were not only positive for MafB-GFP, but also expressed a series of typical podocyte-specific proteins (Figure 13, bottom).
These results indicate that inhibition of TGF-β signals at the stage of differentiation into each nephron site (STEP 3) promotes differentiation into podocytes and inhibits differentiation into other nephron sites, thereby promoting podocyte induction. It suggests increasing efficiency.

Example 5: Selective induction of podocytes from human iPS cells Next, efficient induction of podocytes into podocytes was investigated using nephron progenitor cells induced from human iPS cells. Using the Nephrin-GFP hiPS cell line, we monitored the efficiency of hiPSC induction into podocytes. A three-step stepwise podocyte differentiation induction method established for mouse NPCs was applied to hiPS. An outline is shown in FIG. First, we investigated the concentration and duration of Wnt agonist during the step from NPCs to PA (STEP 1), and then cultured under standard conditions (0.5 μM CHIR99021) until day 9. Podocyte induction efficiency was confirmed by Nephrin-GFP ⁺ cell ratio. The results are shown in FIG. Treatment with 3 μM CHIR99021 for 24 hours was found to be the most efficient in inducing podocytes.

As a result of histological analysis, it was confirmed that NPCs differentiated into PA (LHX1 ⁺ /ECAD ^- ) by CHIR induction for 24 hours, and formed RV (LHX1 ⁺ /ECAD ⁺ ) in 48 hours.
During the differentiation process of PA to RV, SB431542 treatment increased the proportion of podocytes, similar to that in mice. In addition, in the case of hiPSC-derived NPCs, addition of Wnt signal inhibitor (IWR1) and RA in STEP 2 (Step B) also increased the proportion of podocytes. The results are shown in FIG. The combination of these three reagents significantly increased the number of proximal RV cells and increased the final podocyte induction efficiency to over 70%.

In STEP 3 (Step C) after RV formation, SB, IWR1, and RA were all effective in increasing podocyte induction efficiency when administered alone. Furthermore, by combining SB and IWR1, the podocyte induction efficiency was further increased, reaching over 90% (Figure 17).
As mentioned above, by examining and optimizing the culture conditions required for each step, we achieved selective induction of podocytes from hiPSCs-derived NPCs. A schematic of the process is shown in Figure 1b. It has also become clear that this induction method can also be applied to a hiPS cell line (201B7) that has not undergone gene editing. In addition, the yield of podocytes itself was 300,000 podocytes that could be induced from 10,000 hiPSCs, approximately three times the number of normal kidney organoids (Figure 18). Furthermore, in STEP 3, culture could be continued even after the 9th day after the start of culture, and the cells were still alive on the 15th day after the start of culture.

Example 6: Comparison of gene expression profiles of induced podocytes, human podocytes, and cell lines The gene expression profiles of induced podocytes were compared with human adult kidney-derived podocytes and human immortalized podocyte cell lines. As a result of the analysis, induced podocytes showed high expression levels of podocyte-specific genes such as NPHS1, NPHS2, WT1, and SYNPO, and these expression levels were comparable to human adult podocytes (Figure 19) . In the cell line, SYNPO expression was relatively maintained compared to human adult podocytes and induced podocytes, but the expression levels of NPHS1 and NPHS2 were extremely low.

Example 7: Examination of morphological and functional characteristics of induced podocytes As shown in Figure 20, it was confirmed that induced podocytes expressed WT1 in the nucleus and NEPHERIN on the cell border. . Other slit membrane-associated proteins, NEPH1 and PODOCIN, were also localized with NEPHERIN. As a result of transmission electron microscopy, the presence of foot processes on the basal side of podocytes was also confirmed (FIG. 21). Staining with anti-nephrin antibody confirmed that these foot processes expressed nephrin protein (Figure 22). A slit membrane-like structure was also confirmed as filament-like bridges existing between adjacent podocytes (FIG. 21).

Protein expression in induced podocytes was confirmed by Western blotting and compared with hiPSCs, human immortalized podocyte cell lines, and hiPSC-derived renal organoids. As shown in FIG. 23, induced podocytes expressed large amounts of slit membrane-associated proteins, such as NEPHRIN, phosphorylated NEPHRIN (p-NEPHRIN), NEPH1, and PODOCIN. Induced podocytes also expressed SYNAPTOPODIN, a differentiated podocyte marker, and PLA2R, an endogenous antigen of membranous nephropathy, which were higher than in hiPSCs and cell lines. These results suggest that induced podocytes express sufficient amounts of foot process-related proteins.

It was confirmed as follows whether the induced podocytes produced by the method of the present invention could be used for in vitro podocyte damage and toxicity tests. The induced podocytes were treated with puromycin aminonucleoside (PAN) for 48 hours. PAN is a drug that has been used in podocyte damage models in vitro and in vivo. When induced podocytes were treated with PAN, a decrease in podocyte survival rate was confirmed (FIG. 24). This indicates that induced podocytes can be applied to cytotoxicity tests. Further, in a study at the protein level, slit membrane-related proteins expressed on the membrane surface, such as NEPHRIN, p-NEPHRIN, and NEPH1, were significantly reduced by PAN treatment (FIG. 25). Quantifying these slit membrane-associated proteins before and after injury has not been possible with conventional cell lines or podocyte-like cells induced by other protocols. These results indicate that the podocytes produced by the method of the present invention have some of the functional characteristics of podocytes, and can be used as a model for podocyte damage in injury model experiments, screening for therapeutic drug development, and toxicity tests in renal cells. This suggests that it is useful for

Podocyte disorder is the most important pathological condition as a cause of proteinuria, and the development of drugs that protect podocytes in kidney diseases such as diabetic nephropathy and chronic glomerulonephritis is awaited. Podocytes produced by the induction method of the present invention express a large number of podocyte-related proteins, and are useful for screening in the development of drugs that protect podocytes when damaged, i.e., in the field of kidney disease. It is considered to be useful in drug discovery. In addition, induced podocytes can be used as a material for cytotoxicity tests when verifying the toxicity of drugs that may cause kidney damage.
Furthermore, by inducing patient-derived podocytes from iPS cells obtained from patients with congenital nephrotic syndrome, a disease related to podocytes, it can be applied to elucidate the pathology of rare diseases and develop treatments. In addition, it has become possible to gene edit iPS cells using technologies such as CRISPR/CAS9 and TALEN, so it is now possible to establish cell lines in which specific genes are removed or overexpressed, and to inject the original into such cell lines. By applying the induction method of the invention, it is also possible to apply it to an experimental system to examine what role the gene plays in human podocytes.

The above detailed description is merely illustrative of the purpose and subject matter of the invention and is not intended to limit the scope of the appended claims. Various modifications and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings provided herein without departing from the scope of the appended claims.

By the induction method of the present invention, podocytes can be efficiently induced. Furthermore, podocytes induced by the method of the present invention express many podocyte-related proteins and are useful as podocyte mimics.

Claims (22)

A method for inducing differentiation of podocytes from nephron progenitor cells or a population thereof, comprising the following steps:
Step A: culturing nephron progenitor cells or a population thereof in a medium containing a Wnt agonist and a ROCK inhibitor to form renal epithelial progenitor aggregates;
Step B: A step of culturing the renal epithelial precursor aggregate obtained in Step A in a medium containing Fgf to induce differentiation into renal corpuscles, and
Step C: a step of culturing the renal corpuscles obtained in Step B in a medium containing Fgf to induce differentiation into podocytes,
a method including;
Here, at least one of the media in step B or step C contains a TGFβ signal pathway inhibitor. 2. The method of claim 1, wherein the concentration of Wnt agonist in the medium used in step A is greater than 1 μM and less than 10 μM. The method according to claim 1 or 2, wherein the medium in step B and step C further contains a Wnt agonist at a concentration of 1 μM or less. The method according to any one of claims 1 to 3, wherein the culturing in step A is for 12 to 48 hours, and the culturing in step B is for 12 to 48 hours. The method according to claim 4, wherein the culturing in step C is for 2 to 30 days. 6. The method according to any one of claims 1 to 5, wherein the Wnt agonist is CHIR99021 and the TGFβ signal pathway inhibitor is SB431542. The method according to any one of claims 1 to 6, wherein the nephron progenitor cells are nephron progenitor cells derived from mammalian-derived iPS cells. The nephron progenitor cells undergo the following steps:
(a) culturing embryoid bodies induced from mammalian-derived iPS cells in a medium containing a high concentration (concentration A) of a Wnt agonist;
(b) culturing the embryoid bodies in a medium containing Bmp, activin, retinoic acid, and a medium concentration (concentration B) of a Wnt agonist; and (c) culturing the embryoid bodies in a medium containing Fgf and low culturing in a medium containing a Wnt agonist at a concentration (concentration C);
in that order (wherein the concentration of the Wnt agonist is concentration A>concentration B>concentration C, where concentration A is at least 5 times greater than concentration C),
The method according to any one of claims 1 to 7, wherein the iPS cells are derived from mammalian-derived iPS cells. The Wnt agonist in steps (a), (b) and (c) is CHIR99021, and the concentration A and the concentration C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively, and The Bmp in step (b) is Bmp4 and the concentration in the medium is 1 ng/ml to 10 ng/ml, and the concentration of activin in the medium in step (b) is 2.5 to 40 ng/ml. 9. The method of claim 8, wherein the concentration is The method according to any one of claims 7 to 9, wherein the mammalian-derived iPS cells are human-derived iPS cells. The method according to claim 10, wherein the medium in step B and/or step C further contains a Wnt signal inhibitor. The method according to claim 10 or 11, wherein the medium in step B and/or step C further contains retinoic acid (RA) or an RA analog. A medium kit for inducing differentiation of podocytes from nephron progenitor cells derived from human-derived iPS cells, the kit comprising a differentiation-inducing medium and three containers containing the respective components described below :
(1) a first container containing a Wnt agonist and a ROCK inhibitor;
(2) a second container comprising a TGFβ signal pathway inhibitor, a Wnt signal inhibitor, retinoic acid (RA) or an RA analog, and Fgf, and (3) a TGFβ signal pathway inhibitor, a Wnt signal inhibitor, retinoic acid ( RA) or RA analog, and a third container comprising Fgf;
A medium kit comprising: a first, second, and third container each containing a medium for preparing each medium used in the first, second, and third steps of inducing differentiation of podocytes from nephron progenitor cells; A culture medium kit characterized in that it is used for . The culture medium kit according to claim 13, wherein the containers (2) and (3) further contain a Wnt agonist. A method for producing a podocyte cell population from human-derived iPS cells, comprising the following steps:
(i) inducing differentiation of nephron progenitor cells or a population thereof from mammalian-derived iPS cells or a population thereof;
(ii) culturing the nephron progenitor cells or a population thereof obtained in step (i) in a medium containing a Wnt agonist and a ROCK inhibitor to form renal epithelial progenitor aggregates;
(iii) culturing the renal epithelial precursor aggregate obtained in step (ii) in a medium containing Fgf to induce differentiation into renal corpuscles, and
(iv) culturing the renal corpuscles obtained in step (iii) in a medium containing Fgf to induce differentiation into podocytes;
a method including;
Here, at least one of the medium in step (iii) or step (iv) contains a TGFβ signal pathway inhibitor, at least one of the medium in step (iii) or step (iv) contains a Wnt signal inhibitor, and At least one of the media in step (iii) or step (iv) contains retinoic acid (RA) or an RA analog. 16. The method of claim 15, wherein the concentration of Wnt agonist in the medium used in step (ii) is greater than 1 μM and less than 10 μM. 17. The method according to claim 15 or 16, wherein the medium in steps (iii) and (iv) further contains a Wnt agonist at a concentration of 1 μM or less. The method according to any one of claims 15 to 17, wherein the culturing in step (ii) is for 12 hours to 48 hours, and the culturing in step (iii) is for 12 to 48 hours. The method according to any one of claims 15 to 18, wherein the culturing in step (iv) is for 2 to 30 days. 20. The method of any one of claims 15-19, wherein the Wnt agonist is CHIR99021, the TGFβ signal pathway inhibitor is SB431542, and the Wnt signal inhibitor is IWR-1. The step (i) is the following step:
(a) culturing embryoid bodies induced from human iPS cells in a medium containing a high concentration (concentration A) of a Wnt agonist;
(b) culturing the embryoid bodies in a medium containing Bmp, activin, retinoic acid, and a medium concentration (concentration B) of a Wnt agonist; and (c) culturing the embryoid bodies in a medium containing Fgf and low culturing in a medium containing a Wnt agonist at a concentration (concentration C);
in that order (wherein the concentration of the Wnt agonist is concentration A>concentration B>concentration C, where concentration A is at least 5 times greater than concentration C),
The method according to any one of claims 15 to 20. The Wnt agonist in steps (a), (b) and (c) is CHIR99021, and the concentration A and the concentration C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively, and The Bmp in step (b) is Bmp4, the concentration in the medium is 1 ng/ml to 10 ng/ml, and the concentration of activin in the medium in step (b) is 2.5 ng/ml to 40 ng/ml. 22. The method of claim 21, wherein the concentration is mL.