JPWO2019230737A1 - A method for inducing glomerular podocytes and a method for producing podocytes from pluripotent stem cells using the method. - Google Patents

Abstract

An object of the present invention is to provide a method for inducing glomerular podocytes in vitro. More specifically, it is an object of the present invention to provide a method for producing podocytes, which can obtain a cell population having a high composition ratio of podocytes by selectively inducing podocytes. According to the present invention, the following steps: Step A: Culture of Neflon precursor cells or a population thereof in a medium containing a Wnt agonist and a ROCK inhibitor, Step B: Cells or a population thereof obtained in Step A are subjected to Fgf. Step of culturing in a medium containing Fgf and step C: A step of culturing the cells or a population thereof obtained in step B in a medium containing Fgf (here, at least one of the media in step B or step C is TGFβ. Methods have been provided for inducing differentiation of podocytes, including signal pathway inhibitors).

Description

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

Podocytes present in the glomeruli of the kidney play an important role in blood filtration. Podocyte research is an important research theme in terms of health economics, because when podocytes are impaired, proteinuria is exhibited, which eventually progresses to end-stage renal disease and requires dialysis treatment.

In podocyte studies, immortalized cell lines of human podocytes have been used for in vitro analysis (Non-Patent Document 1). However, this cell line has a problem that the expression level of genes and proteins characteristic of podocytes is extremely low, and it is an insufficient material for carrying out research (Non-Patent Document 2).
In the past, it has been reported that podocytes were selectively induced from human iPS cells, but the expression level of NPHS1, which should be a podocyte-specific gene, is 1/5 to 1/5 that of human iPS cells before induction. The expression remains extremely low, 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 referred to as podocytes.

In recent years, including reports by the present inventors, a plurality of studies have been reported in which nephron progenitor cells are induced from human iPS cells to produce renal organoids containing podocytes and tubular cells in vitro (Non-Patent Document 6). ~ 9; Patent Document 2). Renal organoids are a population of multiple cell types, but are cell-autonomous with more purified podocytes for podocyte disorder model experiments and drug screening for the development of therapeutic agents to reduce urinary protein. Verification of (cell-autonomous) effects is required. Therefore, there is a demand for a method for efficiently inducing or producing a cell population consisting of high-purity podocytes.

U.S. Patent Application Published 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

An object of the present invention is to provide a method for inducing glomerular podocytes. Another object of the present invention is to provide a method for producing a podocyte, which can obtain a cell population having a high composition ratio of a podocyte by selectively inducing a podocyte.

As a result of intensive studies to solve the above problems, the present inventors have described the differentiation process from nephron progenitor cells to podocytes in (1) the Mesenchymal-to-epithelical transition (MET) phase (MET). Hereinafter, it may be referred to as a renal epithelial progenitor aggregate (PA) formation stage), (2) renal vesicle (RV) formation stage, and (3) nephron fractionation formation stage (hereinafter referred to as “nephron fractionation stage”). As a result of examining the signals required for induction to podocytes by dividing into three stages (sometimes called the podocyte formation stage), it was clarified that time-specific adjustment of a specific signal is important, and the present invention was completed. did.
The present invention includes the following aspects.
[1] A method for inducing differentiation of podocytes, the following steps:
Step A: A step of culturing Neflon progenitor cells or a population thereof in a medium containing a Wnt agonist (preferably Glycogen Synthase Kinase (GSK) -3β inhibitor, more preferably CHIR99021) and a ROCK inhibitor (preferably Y27632). The cells or cell populations obtained here are referred to as "pre-tubular aggregates (PV)"),
Step B: The step of culturing the cells or a population thereof obtained in Step A in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) (cells or cell populations obtained here are referred to as "steps". Renal corpuscle (RV) "), and
Step C: The step of culturing the cells or a population thereof obtained in Step B in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) (cells or cell populations obtained here are referred to as "steps". Method including, referred to as "podocyte"),
Here, at least one of the media in step B or step C contains a TGFβ signaling pathway inhibitor (preferably SB431542).
[2] 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). The method described.
[3] The medium in steps B and C further comprises 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) [1]. Or the method according to [2].
[4] The culture in the step A is 12 hours to 48 hours (preferably 18 hours to 30 hours, more preferably 21 to 27 hours, still more preferably about 24 hours), and the culture in the step B is 12 hours. The method according to any one of the above [1] to [3], which is time to 48 hours (preferably 18 to 30 hours, more preferably 21 to 27 hours, still more preferably about 24 hours).
[5] The method according to the above [4], wherein the culture in the step C is 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 the above [1] to [5], wherein the Wnt agonist is CHIR99021 and the TGFβ signaling pathway inhibitor is SB431542.
[7] The method according to any one of the above [1] to [6], wherein the nephron progenitor cell is a nephron progenitor cell derived from a mammalian-derived iPS cell.
[8] The method according to the above [7], 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 the above [7] to [9], wherein the medium in the step B and / or the step C further contains retinoic acid (RA) or an RA analog.
[11] The nephron progenitor cells are subjected to the following steps:
(A) A step of culturing an embryoid body derived from human iPS cells in a medium containing a high concentration (concentration A) of a Wnt agonist (preferably CHIR99021).
(B) A step of culturing the embryoid body in a medium containing Bmp (preferably Bmp2, Bmp4 or Bmp7, more preferably Bmp4), activin, retinoic acid, and a medium concentration (concentration B) Wnt agonist. And (c) the step of culturing the embryoid body in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) and a low concentration (concentration C) of Wnt agonist.
(Here, the concentration of the Wnt agonist is concentration A> concentration B> concentration C, and the concentration A is at least 5 times the concentration C).
The method according to any one of the above [7] to [10], which is derived from human iPS cells.
[12] The Wnt agonist in the steps (a), (b) and (c) is CHIR99021, and the concentrations A and C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively. In addition, the Bmp in the 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 the step (b) is 2.5 ng / mL. The method according to [11] above, which has a concentration of ~ 40 ng / mL.

[13] A medium kit for inducing differentiation of podocytes, which comprises a differentiation-inducing medium and three containers containing each of the 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 signaling 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. A second vessel containing Fgf9), and (3) a TGFβ signaling pathway inhibitor (preferably SB431542), a Wnt signal inhibitor (preferably IWR-1), retinoic acid (RA) or RA analog, and Fgf. A third container containing (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9).
[14] The medium kit according to the above [13], 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 cell population of podocytes, wherein the following steps:
(I) A step of inducing differentiation of nephron progenitor cells or a population thereof from a mammalian-derived iPS cell (preferably human iPS cell) or a population thereof.
(Ii) The nephron progenitor cells or the population obtained in step (i) are mixed with a medium containing a Wnt agonist (preferably a GSK-3β inhibitor, more preferably CHIR99021) and a ROCK inhibitor (preferably Y27632). Culturing step (cells or cell populations obtained here are referred to as "pre-tubular aggregate (PA)"),
(Iii) The step of culturing the cells or a population thereof obtained in step (ii) in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) (cells or cell population obtained here). Is referred to as "renal corpuscle (RV)"), and
(Iv) The step of culturing the cells or a population thereof obtained in step (iii) in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) (cells or cell population obtained here). Is referred to as a "podocyte"), a method comprising
Here, at least one of the media in step (iii) or step (iv) contains a TGFβ signal pathway inhibitor (preferably SB431542), and at least one of the media in step (iii) or step (iv) is a Wnt signal. It contains an inhibitor (preferably IWR-1) and at least one of the media in step (iii) or step (iv) contains retinoic acid (RA) or RA analog.
[16] The concentration of the Wnt agonist in the medium used in the 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 comprises 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 according to the above [15] or [16].
[18] The method according to any one of the above [15] to [17], wherein the iPS cells are human-derived iPS cells.
[19] The culture in the step (ii) is 12 hours to 48 hours (preferably 18 hours to 30 hours, more preferably 21 to 27 hours, still more preferably about 24 hours), and the step (ii). The above-mentioned [15] to [18], wherein the culture in the above is 12 hours to 48 hours (preferably 18 hours to 30 hours, more preferably 21 to 27 hours, still more preferably about 24 hours). the method of.
[20] The method according to the above [17], wherein the culture in the step (iv) is 2 to 30 days (preferably about 3 to about 15 days, more preferably 4 to 9 days).
[21] The above-mentioned [15] to [18], wherein the Wnt agonist is CHIR99021, the TGFβ signal pathway inhibitor is SB431542, and the Wnt signal inhibitor is IWR-1. Method.
[22] The step (i) is the following step:
(A) A step of culturing an embryoid body derived from human iPS cells in a medium containing a high concentration (concentration A) of a Wnt agonist (preferably CHIR99021).
(B) A step of culturing the embryoid body in a medium containing Bmp (preferably Bmp2, Bmp4 or Bmp7, more preferably Bmp4), activin, retinoic acid, and a medium concentration (concentration B) Wnt agonist. And (c) the step of culturing the embryoid body in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) and a low concentration (concentration C) of Wnt agonist.
(Here, the concentration of the Wnt agonist is concentration A> concentration B> concentration C, and the concentration A is at least 5 times the concentration C).
The method according to any one of the above [15] to [21].
[23] The Wnt agonist in the steps (a), (b) and (c) is CHIR99021, and the concentrations A and C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively. In addition, the Bmp in the 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 the step (b) is 2.5 ng / mL. The method according to [20] above, which has a concentration of ~ 40 ng / mL.

[24] A method for evaluating the effect or effect of a test substance on a podocyte, which is described in any one of the above [1] to [12] and [15] to [23] in the presence of the test substance. The cell population of podocytes prepared by the above method was cultured, and the following items related to the cells: cell proliferation, cell survival, cell damage, 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 transduction signals and assessing the effect of the test substance on podocytes.
[25] A method for screening a substance for treating or ameliorating a podocyte disorder, which was prepared by the method according to (x) any one of the above [1] to [12] and [15] to [23]. The step of culturing a podocyte cell population in the presence of a drug and test substance exhibiting podocyte damage, and (y) podocyte survival, proliferation or reduction of damage, changes in protein expression level, and changes in gene expression level. A step of measuring at least one item selected from the group consisting of, and a method of screening for candidate substances for treating or ameliorating a podocyte disorder, including.
Here, the culture of the podocyte cell population in step (x) may be any of the following:
Cultivate the podocyte cell population in the presence of (x-1) podocyte-damaging drug and test substance at the same time, or cultivate the podocyte cell population in the presence of (x-1) podocyte-damaging drug. Then, the damaged podocyte cell population is cultured in the presence of the test substance.
[26] The screening method according to the above [25], wherein the drug exhibiting podocyte damage is at least one drug selected from the group consisting of puromycin aminonucleoside, doxorubicin, angiotensin II, and an anti-podocyte antibody.

[27] A method of treating or preventing kidney and / or glomerular disease.
(I) A podocyte cell population prepared by the method according to 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. Transplanting (where the podocyte cell population is optionally transplanted with stromal cells and / or vascular endothelial cells and / or podocytes), and (II) in the transplanted podocyte cell population. At least some of the cells of the kidney tissue migrate to and stay in the glomerular body of the renal tissue, thereby regenerating the renal tissue and treating kidney and / or glomerular disease.
How to include.
[28] The renal and / or glomerulonephropathy is a condition associated with podocyte disorder, nephrotic syndrome, nephrosclerosis, diabetic nephropathy, chronic glomerulonephritis, or a combination thereof, or a complication of those diseases. The method according to the above [27].
[29] The method according to [27] or [28] above, wherein the cell population of the podocyte is transplanted as a part of a human iPS cell or a renal organoid induced to differentiate from the population.

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

It is a figure which showed the outline example of the protocol of the induction of a podocyte of this invention. The upper figure (a) is an example of a method of inducing podocytes using mouse nephron progenitor cells, and the lower figure (b) is an example of a method of inducing podocytes using nephron progenitor cells derived from human iPS cells. Shown. CHIR: CHIR99021; Y: Y27632; SB: SB431542; F: Fgf9; RA: retinoic acid. It is a figure which showed the gene expression of each cell of Six2-GFP negative cell, Six2-GFP positive nephron progenitor cell (NPCs), and nephron progenitor cell (NPCs) selected by an antibody. It is a figure which showed the outline of the experiment which confirmed the influence of the Wnt agonist (CHIR99021) using the nephron progenitor cells (NPCs) prepared in Example 1. It is a figure which showed the result of treating the Wnt agonist (CHIR99021) at the respective concentration and time in the induction of podocytes using the nephron progenitor cells (NPCs) prepared in Example 1. It is a micrograph on the 6th day. In the induction of podocytes using nephron progenitor cells (NPCs), the presence of MafB-GFP-positive podocytes was confirmed on the 6th day when CHIR99021 was treated at a concentration of 3 μM or 5 μM for 24 hours or 48 hours. The above figure shows the observation results with visible light and fluorescence. The figure below is a graph of the results. Time series (0 hours, 0 hours, It is the result confirmed in 24 hours, 48 hours). The results of confirming the gene expression of Six2, Lhx1, Pax8, and Cdh1 in the induction process are shown. This is the result of confirming the effect of growth factors or inhibitors in the process of inducing differentiation of renal epithelial precursor aggregates (PA) to renal corpuscles (RV) by microscopic observation. a is CHIR99021 (2 μM) and SB431542, and b is IWR-1, Activin A, RA and BMS493. The upper row shows the results of observing cell growth by visible light, and the lower row shows the results of observing MafB-GFP positive cells by fluorescence. This is the result of confirming the effect of growth factors or inhibitors in the process of inducing differentiation of renal epithelial precursor aggregates (PA) into renal corpuscles (RV). The percentage of MafB-GFP positive cells (a) and the number of podocytes (b) are shown. This is the result of examining gene expression by SB431542 treatment in the renal corpuscle (RV) stage (48 hours). This is the result of confirming the ratio and number of Wt1-positive proximal renal corpuscle (RV) cells treated with SB431542. We confirmed the effects of each factor (CHIR99021 (2 μM), IWR-1, activin, SB431542, retinoic acid (RA), and BMS493) on the differentiation into the podocyte lineage in STEP3 after induction of proximal corpuscle (RV). Results. A: The upper row is the result of observing cell growth by visible light, and the lower row is the result of observing MafB-GFP positive cells by fluorescence. B: MafB-GFP positive cells (podocytes) in the cell population. Shows the percentage. This is the result of confirming the effect of SB431542 on the cell number of each nephron (podocyte, PT, and DT) in STEP3 after induction of proximal renal corpuscle (RV). This is the result of confirming the expression of the podocyte-specific protein and the expression of other tubular markers in the podocytes prepared by the method of the present invention. It is a schematic diagram of an example when the three-step stepwise podocyte differentiation induction method established in mouse NPCs is applied to human iPS cells (hiPS). It is a figure showing the result of confirming the influence of the Wnt agonist (CHIR99021) in STEP1 in the induction of podocytes using nephron progenitor cells (NPCs) derived from human iPS cells (hiPS). It is a figure which showed the result of confirming the influence of addition of various factors in STEP2 in the induction of podocytes using nephron progenitor cells (NPCs) derived from human iPS cells (hiPS). It is a figure which showed the result of confirming the influence of addition of various factors in STEP3 in the induction of podocytes using nephron progenitor cells (NPCs) derived from human iPS cells (hiPS). It is a result of comparing the podocyte induced by the method of the present invention with the podocyte of the renal organoid. The figure on the left compares the ratio, and the figure on the right compares the number of podocytes. It is a figure which compared the expression of the podocyte-specific gene in the induction podocyte, the human adult kidney-derived podocyte, the human iPS cell, and the immortalized podocyte cell line. It is a figure which shows the expression site of WT1, NEPHERIN, NEPH1, and PODOCIN in an induction podocyte. It is a photograph which observed the induction podocyte with a transmission electron microscope. The arrows in the left figure indicate the foot processes located in the lateral basal region. The arrows in the right figure indicate the slit film-like structure (filament-like crosslinks) existing between podocytes. It is another photograph which observed the induction podocyte with a transmission electron microscope. The arrow points to the NEPHRIN molecule. It is the result of analyzing the protein expression of the induced podocyte by Western blotting. It shows the survival rate when the induced podocytes are treated with puromycin aminonucleoside (PAN). It is the result of Western blot analysis of the protein expression when the induced podocyte was treated with puromycin aminonucleoside (PAN).

Hereinafter, the present invention will be described with preferred methods and materials that can be used in the practice of the present invention, using exemplary embodiments as examples, but the present invention is not limited to the embodiments described below. Unless otherwise specified in the text, all technical terms and scientific terms used in the present specification have the same meanings as generally understood by those skilled in the art to which the present invention belongs. Also, any material and method equivalent to or similar to that described herein can be used similarly in the practice of the present invention. In addition, all publications and patents cited herein in connection with the invention described herein are described herein, for example, as indicating methods, materials, etc. that can be used in the present invention. It constitutes a part.

In the present specification, when the expressions "X to Y" are used, they are used to mean that X is included as the upper limit and Y is included as the upper limit, or that X is included as the lower limit and Y is included as the upper limit. As used herein, "and / or" is used to include either one or both. Further, in the present specification, "about" is used in the sense of allowing ± 10%.

The kidney is generated by the interaction of nephron progenitor cells (NPCs), which are precursors of glomerular podocytes and tubules, ureteral buds, which are precursors of collecting ducts and ureters, and stromal cells. Neflon progenitor cells undergo epithelial-mesenchymal transition (MET) and develop into epithelial nephron structures, including podocytes, proximal tubules (PT), and distal convoluted tubules (DT). Nephron progenitor cells form renal progenitor aggregates (PAs) by Wnt signaling from ureteral buds and then differentiate into proximal and distal polar corpuscles (RVs). The renal corpuscle (RV) extends along the proximal distal axis and differentiates into the nephron epithelium via the S-shape. Progenitor cells of podocytes are located in the WT1-positive proximal domain of the renal corpuscle (RV) and the Wt1-positive proximal domain of the S-shape, and eventually differentiate into MafB-positive / Nephrin-positive mature podocytes. ..
It has been suggested that Wnt and Notch signals play a role in the process of proximal and distal axis formation of nephrons, but it is unclear what signals are involved in nephron fractionation, especially podocyte differentiation. Met.

The present inventors did not go through the nephron progenitor cells capable of differentiating into the entire nephron fraction in the induction method in the conventional reports regarding the induction of podocytes, and along the process of differentiation of nephron progenitor cells into podocytes. We focused on the fact that we have not been able to make a step-by-step study. And, 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 are not guaranteed. It happened.

Therefore, the present inventors derived nephron progenitor cells capable of differentiating into a total nephron fraction from human iPS cells based on the conventional reports of the present inventors (Non-Patent Document 6), and then nephrons. The differentiation process from progenitor cells to podocytes is divided into three stages: (1) mesenchymal epithelial conversion induction (MET) stage, (2) renal vesicle (RV) formation stage, and (3) nephron fractionation stage. We examined the signals required for induction to podocytes and found that time-specific regulation of CHIR99021, SB431542, IWR-1, retinoic acid, and Fgf9 is important. Podocyte derived in accordance with the present invention, 10 ⁴ times the human iPS cells in 10 ⁵ times the height of the immortalized cell lines are expressing NPHS1 (Nephrin), iPS cells and immortalized in other podocyte related protein It showed extremely high expression levels compared to cell lines. Furthermore, the inventors separated podocytes from the adult human kidney and compared the induced podocytes with the real podocytes, and the induced podocytes made podocyte-related proteins such as NPHS1 and NPHS2 (Podocin) almost the same as the in vivo podocytes. It was confirmed that it was expressed at the same level. That is, the induced podocytes according to the present invention show almost the same quality as the real podocytes in vivo at the gene expression level, which proves the validity of the induction method of the present invention.

One aspect of the present invention is a method of inducing podocytes from nephron progenitor cells, in three steps: induction of nephron progenitor cells to renal epithelial progenitor aggregates (PA), PA to renal corpuscle (RV). ), And the method includes induction from RV to podocytes.
Another aspect of the present invention is a method of selectively inducing glomerular podocytes from nephron progenitor cells using the above three steps.
Another aspect of the present invention is a method for producing glomerular podocytes from nephron progenitor cells using the above three steps.
Another aspect of the present invention is a method for producing podocytes from mammalian-derived iPS cells (preferably human iPS cells), the step of inducing nephron progenitor cells from iPS cells, and the above 3). It is a method for producing podocytes, which is a combination of steps of inducing podocytes from nephron progenitor cells including one step.
Further, one aspect of the present invention is a medium kit for inducing differentiation of podocytes from nephron progenitor cells, which includes a container containing various substances used in the above three steps and a medium.
Yet another aspect of the present invention is to provide an experimental model of a podocyte using a podocyte derived by any of the above methods.
Yet another aspect of the present invention is a method for evaluating a substance having cytotoxicity to a podocyte using a podocyte induced by any of the above methods.
Another aspect of the present invention is a method for screening a substance for improving or treating a podocyte disorder or a disease related thereto, using a podocyte induced by any of the above methods.
Yet another aspect of the invention comprises transplanting a podocyte induced by any of the above methods into a subject suffering from a disease of the kidney and / or glomerulus. It is a method of treating or preventing the disease of.

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

Nephron progenitor cells As the nephron progenitor cells used in the method of the present invention, either those prepared from a living body or those derived from pluripotent stem cells can be used, but those derived from pluripotent stem cells are preferable. 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 traits under specific conditions, or by cryopreserving them. Induction of nephron progenitor cells from pluripotent stem cells can be performed with reference to known methods. For example, it can be prepared with reference to the reports by the present inventors (Non-Patent Document 6; Patent Document 2). These documents are part of this specification by reference.
Although not limited to this, for example, the following steps can be mentioned.
(A) A step of culturing an embryoid body derived from pluripotent stem cells in a medium containing a high concentration (concentration A) of a Wnt agonist (preferably CHIR99021).
(B) A step of culturing the embryoid body in a medium containing Bmp (preferably Bmp2, Bmp4 or Bmp7, more preferably Bmp4), activin, retinoic acid, and a medium concentration (concentration B) Wnt agonist. And (c) the step of culturing the embryoid body in a medium containing Fgf (preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9) and a low concentration (concentration C) of Wnt agonist.
(Here, the concentration of the Wnt agonist is concentration A> concentration B> concentration C, and the concentration A is at least 5 times the concentration C) to prepare Neflon precursor cells. can.

..
The embryoid body used in the above step (a) can be prepared by culturing pluripotent stem cells (for example, ES cells and iPS cells) in an arbitrary medium, preferably a serum-free medium. For example, when human pluripotent stem cells are used, it is preferable to use human iPS cells treated with Bmp4 and a Rock inhibitor (Y27632) (with additional Fgf if necessary). The embryoid body is prepared, and more preferably, the embryoid body treated with actibin and Fgf is used. 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 the Rock inhibitor (Y27632) in the medium is, for example, 1 to 1. 100 ng / mL, preferably 5 to 20 ng / mL can be raised, the concentration of Fgf in the medium can be raised, for example, 0 to 20 ng / mL, and the concentration of activin in the medium can be raised, for example, 0. .1 to 5 ng / mL, preferably 0.5 to 1 ng / mL can be given. Treatment with Bmp4 and Rock inhibitor (Y27632), Fgf can be carried out, for example, for 1-2 days, preferably 1 day, and treatment for activin and Fgf can be carried out, for example, for 1-4 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 into somatic cells in the form of DNA or protein. , 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). Reprogramming factors are genes that are specifically expressed in ES cells, their gene products or their non-coding RNA, genes that play an important role in maintaining undifferentiated ES cells, their gene products or their non-coding RNA, or It may be composed of a low molecular weight compound. Examples of genes included in the reprogramming factor are Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-MYC, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras. , ECAT15-2, Tcl1, beta-catenin, Lin28b, All1, All4, Esrrb, Nr5a2, Tbx3, Glis1 and the like. These reprogramming factors may be used alone or in combination. When the c-MYC gene is introduced into a somatic cell and used as a reprogramming factor, an introduction method in which the introduced c-MYC gene is unlikely to be integrated into the chromosome of the target cell after the creation of iPS cells is used. Is preferable, and examples thereof include, but are not limited to, introduction using a Sendai virus vector or an episomal vector.

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 the Wnt agonist in the medium used in each step is concentration A> concentration B> concentration C, and the concentration A is at least 5 times the concentration C, but preferably the concentration A is at least 2 times the concentration B. And the concentration B is at least twice the concentration C, more preferably the concentration A is at least three times the concentration B, and the concentration B is at least three times the concentration C. The concentration C of the Wnt agonist in the 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, 0.1 to 3.0 μM is preferable. Can be raised from 0.5 to 2.0 μM.
For example, when CHIR99021 is used, the concentration A is 6 to 20 μM, preferably 7 as a combination of Wnt agonist concentrations A, B and C in steps (a), (b) and (c). The concentration B is selected from 2 to 6 μM, preferably 2 to 4.5 μM, more preferably about 3 μM, and the concentration C is 0.5 to 2 μM, preferably 0.5 to 2 μM. Can be a combination selected from 0.7 to 1.5 μM, more preferably about 1 μM.
As the Wnt agonist used, Wnt agonis that can be used in the method of the present invention described below can be used.

As the Bmp used in the step (b), any of the above-mentioned Bmps can be used, and the concentration of each Bmp compound used can be appropriately selected according to the purpose of use. For example, when Bmp4 is used, Bmp4 of any origin can be used, but human Bmp4 is preferable, 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. It can be up to 30 ng / mL, preferably 1-10 ng / mL. When another Bmp compound is used, a concentration capable of exerting the same effect as that obtained when Bmp4 is used can be appropriately selected.

As the activin used in the step (b), activin of any origin can be used, but human activin A is preferable. The concentration in the medium is not particularly limited as long as the effect of inducing differentiation can be obtained, and for example, 2.5 to 40 ng / mL, preferably 7.5 to 15 ng / mL can be given.
The concentration of retinoic acid used in step (b) in the medium is not particularly limited as long as the effect of inducing differentiation can be obtained, but is, for example, 0.001 to 1 μM, preferably 0.01 to 0.3 μM. be able to.

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

The preferred combination in steps (a) to (c) is that the Wnt agonist is CHIR99021 and the concentrations A and C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively. The Bmp in the 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 the step (b) is 2.5 to 40 ng / mL. It is a method for inducing Neflon precursor cells, which is the concentration of.

The induced pluripotent stem (iPS) cells may be of mammalian origin, and the mammals include, for example, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, cows, horses, goats, monkeys or Humans and the like can be mentioned, preferably mice or humans.

Nephron progenitor cells prepared from living organisms or derived from mammalian pluripotent stem cells can optionally be further subjected to a cell selection step. Sorting can be performed using a cell sorter by, for example, at least one of the markers selected from the group consisting of ITGA8, ROBO2, PDGFRa, PDGFRb, CXCR4, and NGFR, or any combination thereof.

Induction of podocytes Induction of podocytes from the nephron progenitor cells of the present invention consists of three steps A to C.
Step A: A step of culturing a nephron progenitor cell 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) (cells obtained here). Alternatively, the cell population is referred to as a "pre-tubular 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). Steps (cells or cell populations obtained here are referred to as "renal vesicles (RVs)"), 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). Steps (cells or cell populations obtained here are referred to as "podocytes"),
However, if the medium in step B contains a TGFβ signal pathway inhibitor, the medium in step C may not contain the TGFβ signal pathway inhibitor, and if the medium in step C contains a TGFβ signal pathway inhibitor, the step The medium in B may be free of TGFβ signaling pathway inhibitors.

Step A (STEP 1) : Induction of NPCs into Renal Epithelial Progenitor Aggregates (PA) Step A is a step of culturing nephron progenitor cells or a population thereof in a medium containing a Wnt agonist and a ROCK inhibitor (preferably Y27632). Is. In the present specification, the cells or cell population obtained in this step are referred to as "pre-tubular aggregate (PA)".
Wnt agonists are defined as agents that activate TCF / LEF-mediated transcription in cells. Wnt agonists are therefore selected from true Wnt agonists that bind to and activate Frizzled receptor family members, including all of the Wnt family proteins, inhibitors of intracellular β-catenin degradation and TCF / LEF activators. NS. 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 of the Glycogen Synthase Kinase (GSK) -3β protein (eg, phosphorylation ability to β-catenin), for example, CHIR99021 (CAS No .: 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 agonists used in the present invention are preferably CHIR99021, SB216763, BIO, R-Spondin1, A 1070722, Lithium carbonate, 3F8, SB 415286, TDZD 8, TWS 119, TCS 2002, Wnt3, Wnt3a, Wnt9. More preferably, it is CHIR99021 or SB216763, and even more preferably CHIR99021.

When CHIR99021 is used as the 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 about 3 μM, preferably about 8 μM, more preferably about 6 μM, still 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 step A within this range, NPCs can be efficiently differentiated into PA. When another Wnt agonist is used instead of CHIR99021 in step A, a concentration showing the same effect can be set with reference to CHIR99021.

The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK). HA 1100 chloride can be used, preferably Y27632 or Fasudil chloride, more preferably Y27632. When Y27632 is used, 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. When another Rock inhibitor is used instead of Y27632 in step A, a concentration showing the same effect can be set with reference to Y27632.

The culture time of 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. It takes about 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 Corpuscle (RV) Step B is the step of culturing PA cells or a population thereof in a medium containing Fgf and optionally a TGFβ signaling pathway inhibitor. As used herein, the cells or cell populations obtained in this step are referred to as "renal vesicles".
The TGFβ signal pathway inhibitor contained in at least one of step B or step C is defined as a substance that inhibits signal transduction from binding of TGFβ to a receptor to SMAD, and is, for example, a receptor for TGFβ. Numerous substances have been reported, including substances that inhibit binding to the ALK family and substances that inhibit the 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 an inducing effect on desired cells can be obtained, but is an ALK inhibitor, SB431542 (CAS No .: 301836-41). −9) or A83-01 (CAS number: 909910-43-6), D4476, GW788388, LY364497, R268712, RepSox, SB505124, SB525334, or SD208, preferably SB431542 or A83-01, with SB431542 More preferred.
When SB431542 is used as the TGFβ signal pathway inhibitor in step B, the concentration of SB431542 in the medium of step B varies depending on the origin of the cells used and the like, but is usually 1 μM to 500 μM, preferably 2 μM to 100 μM, more preferably. Is 3 μM to 50 μM, and is particularly preferably about 25 μM when mouse-derived Neflon progenitor cells are used in step A, and about 25 μM when human pluripotent stem cell-derived Neflon progenitor 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 a proximal polarity. When another component is used instead of SB431542 in step B, a concentration showing the same effect can be set with reference to SB431542.

Fibroblast growth factor (Fgf) can include, for example, Fgf2, Fgf4, Fgf7, Fgf9, or Fgf20, which are prepared by referring to a method known per se based on known amino acid sequence information. Also, a recombinant human Fgf protein purchased from R & D Systems or the like can 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 the culture conditions and the like, 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 further preferably about 10 ng / mL. .. When another Fgf is used instead of Fgf9, a concentration showing the same effect can be set with reference to Fgf9.

The medium of step B may further contain a Wnt agonist at a concentration of about 1 μM or less. The Wnt agonist that can be used is the same as the Wnt agonist described in step A, but is preferably a GSK-3β inhibitor, more preferably CHIR99021.
When CHIR99021 is used as the 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 another Wnt agonist is used instead of CHIR99021, a concentration showing the same effect can be set with reference to CHIR99021.

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

When IWR-1 is used as the Wnt signal inhibitor in step B, the concentration of IWR-1 in the medium of 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, still more preferably about 1 μM to about 2 μM. When another Wnt signal inhibitor is used instead of IWR-1, a concentration showing the same effect can be set with reference to IWR-1.

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

In step B, retinoic acid analog can be used instead of retinoic acid. Examples of the retinoic acid analog include AGN193109, AM580, AM80, BMS453, BMS195614, AC 261066, AC55649, and Isotretinoin, and AGN193109 is particularly preferable. When RA analog is used instead of RA in step B, a concentration showing the same effect can be set with reference to RA.

The culture time of 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, still more preferably about. 24 hours. By setting the culture time in step B within this range, PA can be efficiently differentiated into RV.

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

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, and more preferably Fgf9. Further, as the concentration of Fgf in the medium in step C, the same concentration as that described in step B can be used.

The medium of step C may further contain a Wnt agonist at a concentration of about 1 μM or less. The Wnt agonist that can be used is the same as the Wnt agonist described in step A, but is preferably a GSK-3β inhibitor, more preferably CHIR99021.
When CHIR99021 is used as the 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 another Wnt agonist is used instead of CHIR99021, a concentration showing the same effect can be set with reference to CHIR99021.

When podocytes are induced from human iPS cell-derived nephron progenitor cells using PA derived 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 the Wnt signal inhibitor to both the medium and the medium of step C.
The Wnt signal inhibitor used in step C is the same as the Wnt signal inhibitor described in step B, but is preferably IWR-1. When the Wnt signal inhibitor is added to the medium in step C, its concentration is the same as that described in step B.

When using RV derived from human iPS cell-derived nephron progenitor cells by Step A and Step B, retinoic acid (RA) or RA analog may be added to the medium of 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 when RA or RA analog is added to the medium in step C, the concentration thereof is the one described in step B. Is similar to.

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

As described above, when human iPS cell-derived nephron progenitor cells are used, the present invention describes the following steps:
Step A: A step of culturing a nephron progenitor cell 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 obtained in Step A or a population thereof are subjected to TGFβ signaling pathway inhibitors (preferably SB431542), Wnt signaling inhibitors (preferably IWR-1), RA or RA analogs, and Fgf (preferably, Fgf). A step of culturing in a medium containing Fgf2, Fgf9 or Fgf20, more preferably Fgf9), and
Step C: The cells obtained in Step B or a population thereof are subjected to TGFβ signaling pathway inhibitors (preferably SB431542), Wnt signaling inhibitors (preferably IWR-1), RA or RA analogs, and Fgf (preferably, Fgf). A step of culturing in a medium containing Fgf2, Fgf9 or Fgf20, more preferably Fgf9).
However,
When the medium in step B contains a TGFβ signal pathway inhibitor, the medium in step C may not contain the TGFβ signal pathway inhibitor, and when the medium in step C contains a TGFβ signal pathway inhibitor, in step B. The medium may be free of TGFβ signaling pathway inhibitors and may not contain.
When the medium in step B contains a Wnt signal inhibitor, the medium in step C does not have to contain a Wnt signal inhibitor, and when the medium in step C contains a Wnt signal inhibitor, the medium in step B contains Wnt. It does not have to contain signal inhibitors,
When 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 of inducing differentiation of podocytes, including.

The medium used in the present invention can be prepared using the medium used for culturing animal cells as the basal medium. Examples of the basal medium are, but are not limited to, DMEM (Dalbeco's Modified Eagle's Medium), DMEM / F12 Medium, GMEM (Grasgow MEM) Medium, Ham's F12 Medium, IMDM ( Iscob-modified Dalveco medium), αMEM (Eagle's minimum essential medium α-modified type), 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 be, for example, albumin, ITS (insulin, transferase, selenium), N-2 supplement (Thermo Fisher Scientific), B-27 (registered trademark) supplement minus vitamin A (Thermo Fisher Scientific), if necessary. ), 2-Mercaptoethanol, 1-thioglycerol, amino acids, L-glutamine, non-essential amino acids, ascorbic acid, at least one or more media additives such as antibiotics including penicillin streptomycin may also be contained.

Medium Kit One aspect of the present invention is a medium kit for deriving podocytes from nephron progenitor cells. The medium kit consists of a differentiation-inducing medium and three media containing each of the following components (1) to (3).
(1) Wnt agonist and ROCK inhibitor, (2) TGFβ signaling pathway inhibitor and Fgf, and (3) TGFβ signaling pathway inhibitor and Fgf.
The above (2) may further contain a Wnt signal inhibitor and a retinoic acid (RA) or RA analog, and the above (3) further comprises a Wnt signal inhibitor and a retinoic acid (RA) or RA analog. Although it may be contained, it is preferable that the nephron progenitor cells contain these components when they are derived from human iPS.
It is preferable that the components (1) to (3) above are stored in separate containers. Therefore, the medium kit according to one aspect of the present invention includes a differentiation-inducing medium and a kit consisting of three containers containing the respective components (1) to (3) above.

The Wnt agonist, ROCK inhibitor, TGFβ signaling pathway inhibitor, Fgf, Wnt signaling inhibitor, and retinoic acid (RA) or RA analog used in the medium kit of the present invention are described in the method for inducing podocytes of the present invention. It is the same ingredient as the one. The Wnt agonist is preferably a GSK-3β inhibitor, more preferably CHIR99021, the ROCK inhibitor is preferably Y27632, the TGFβ signaling pathway inhibitor is preferably SB431542, and Fgf is preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9, and the Wnt signal inhibitor is preferably IWR-1.
The Wnt agonist, ROCK inhibitor, TGFβ signaling pathway inhibitor, Fgf, Wnt signal inhibitor, and retinoic acid (RA) or RA analog, which are the above components, have concentrations used in the method for inducing podocytes of the present invention. It can be added to the medium and used as described above.

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

Production of Podocytes One aspect of the present invention is a method for producing a cell population of podocytes from mammalian-derived iPS cells, which comprises the above-mentioned three-step induction step. Therefore, the present invention includes the following aspects.
(I) A step of inducing differentiation of nephron progenitor cells or a population thereof from a mammalian-derived iPS cell (preferably human iPS cell) or a population thereof.
(Ii) A step of 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) A step of culturing the cells or a population thereof obtained in step (ii) in a medium containing a TGFβ signaling pathway inhibitor, a Wnt signaling pathway inhibitor, Fgf, and optionally retinoic acid (RA) or RA analog. as well as,
(Iv) A step of culturing cells or a population thereof obtained in step (iii) in a medium containing a TGFβ signaling pathway inhibitor Wnt signaling inhibitor, Fgf, and optionally retinoic acid (RA) or RA analog.
However,
If the medium in step (iii) contains a TGFβ signal pathway inhibitor, the medium in step C may not contain the TGFβ signal pathway inhibitor, and if the medium in step C contains a TGFβ signal pathway inhibitor, step C. The medium in B may be free of TGFβ signaling pathway inhibitors and may not contain.
If the medium in step (iii) contains a Wnt signal inhibitor, the medium in step (iv) does not have to contain a Wnt signal inhibitor, and if the medium in step (iv) contains a Wnt signal inhibitor. The medium in step (iii) may be free of Wnt signaling inhibitors.
If the medium in step (iii) contains RA or RA analogs, the medium in step (iv) may not contain RA or RA analogs.
Is a method for producing a cell population of podocytes from iPS cells.

The Wnt agonist, ROCK inhibitor, TGFβ signaling pathway inhibitor, Wnt signal inhibitor, Fgf, and retinoic acid (RA) or RA analog used in the above production method are those described in the method for inducing podocytes 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, and the Fgf is preferably Fgf2, Fgf9 or Fgf20, more preferably Fgf9, a TGFβ signaling pathway. The inhibitor is preferably SB431542 and the Wnt signal inhibitor is preferably IWR-1.
Concentrations of each of the above components, Wnt agonist, ROCK inhibitor, TGFβ signaling pathway inhibitor, Fgf, Wnt signal inhibitor, and retinoic acid (RA) or RA analog in the medium, and the medium containing these components. As for the culture period, the description in the method for inducing podocytes of the present invention can be applied as it is.
Further, in step (i) in the above-mentioned production method, the description regarding the induction of nephron progenitor cells from iPS cells described in connection with the method for inducing podocytes of the present invention can be applied as it is.

The cell population of podocytes produced using the method for producing podocytes of the present invention means a cell population composed of cells that are substantially podocytes, and specifically, of all cells constituting the cell population. About 40% or more, preferably about 50% or more, more preferably about 60% or more, still more preferably about 70% or more, even more preferably about 80% or more, most preferably about 90% or more are composed of podocytes. 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. Cell markers include, but are not limited to, NPHS1, NPHS2, WT1, MAFB, or SYNPO. The detection of the composition ratio can be measured using, for example, a cell sorter or immunostaining.

The podocytes produced using the method for producing podocytes of the present invention are characterized by having similar expression with respect to the expression of NPHS1, NPHS2, WT1, MAFB and SYNPO as compared with genuine podocytes (podocytes derived from human adults). .. "Similar expression with respect to the expression of NPHS1, NPHS2, WT1, MAFB and SYNPO" refers to genes for at least two, preferably at least three, more preferably four, and even more preferably all of these five genes. And / or mean that they are equally expressed with respect to protein expression. Equal expression is about 40% or more, preferably about 50% or more, more preferably about 60% or more, still more preferably about 70% or more, more than the expression of a human adult-derived podocyte gene and / or protein. More preferably, it means that the expression is about 80% or more, and most preferably about 90% or more.

Evaluation or Screening Method Using Podocytes One aspect of the present invention is a cell model for evaluation or screening using podocytes produced using the above-mentioned podocyte induction method or production method, and such It is an evaluation or screening method using a cell model.
The evaluation method of the present invention is a method including culturing a podocyte or a cell population thereof produced by using the above-mentioned podocyte induction method or production method together with a target test substance and confirming the effect on the podocyte cell. be. The effects on podocyte cells are not limited to, for example, cell proliferation, cell survival, cell damage, protein expression level, gene expression level, albumin uptake ability, albumin filtration ability, autophagy activity or intracellular transmission. It can be evaluated 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 used is not particularly limited, and examples thereof include low molecular weight compounds, medium molecular weight compounds, high molecular weight compounds, amino acids, peptides, proteins, and natural extracts.
The measurement of the above evaluation items can be carried out by appropriately referring to a known method. Reagents and kits for measuring these evaluation items are also commercially available, and they can be used without limitation.
By using the evaluation method of the present invention, for example, the toxicity of a drug that may cause renal damage can be evaluated without limitation.

Examples of the screening method of the present invention include a method of screening a substance for treating or ameliorating a podocyte disorder. Candidate substances can be screened by performing the following steps using the podocytes produced by the above-mentioned method for inducing podocytes or the method for producing them or the cell population thereof.
(X) A step of culturing in the presence of a drug and a test substance exhibiting podocyte damage, and (y) a step of measuring the survival or proliferation of podocyte cells.
In the culture of the podocyte cell population in the step (x), the podocyte cell population may be cultured in an environment in which (x-1) a drug exhibiting podocyte damage and a test substance are present at the same time, or (x-1). ) Podocyte populations may be cultivated in the presence of agents exhibiting podocyte damage, followed by podocyte populations in the presence of test substances. In the latter case, the test substance may be added after removing the drug exhibiting podocyte damage, or the test substance may be added without removing the drug.
Drugs exhibiting podocyte damage include, but are not limited to, anti-podocyte antibodies such as puromycin aminonucleoside, doxorubicin, angiotensin II, and anti-nephrin antibody, preferably puromycin aminonucleoside. Or doxorubicin.
The test substance is not particularly limited, and examples thereof include low molecular weight compounds, medium molecular weight compounds, high molecular weight compounds, amino acids, peptides, proteins, and natural extracts.
By using the screening method of the present invention, for example, it is possible to search for a drug that acts protectively against podocytes, but is not limited to this.

It is also possible to prepare a podocyte cell population from iPS cells derived from a patient having a podocyte-related disease by using the method for inducing or producing podocytes of the present invention. By using the podocyte cell population thus prepared, it is possible to evaluate and screen a drug for treating a podocyte-related disease. Such an evaluation method or a screening method is also a part of the present invention.

Treatment of Kidney and Glomerular Diseases One aspect of the present invention is a method of treating or preventing kidney and / or glomerular diseases using podocytes produced using the above-mentioned podocyte induction method or production method. .. Therefore, the present invention presents (I) transplanting a podocyte cell population prepared by the above method for inducing or producing podocytes into a part of kidney tissue of a diseased subject, and (II) transplanting podocyte cells. At least some cells in the population move into and stay in at least some of the glomerular body of renal tissue, thereby regenerating kidney tissue and treating kidney and / or glomerular disease. It is also a method including ,.

The subject affected by the disease is not particularly limited as long as it is a mammal, and examples thereof 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 part of the kidney tissue in (I) above, the podocyte cell population can also be transplanted together with other cells or cell scaffolds that assist podocyte transplantation. Other cells include, but are not limited to, stromal cells and vascular endothelial cells. As the cell scaffold, known or commercially available ones can be appropriately used, and examples thereof include scaffolds composed of biocompatible substances (also referred to as biomaterials). 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, poly. Hydrohydroxyalkanoates, dextran, PHA, collagen, chitosan, alginates, and other biocompatible polymers can be mentioned.

The podocytes induced by the present invention can also be transplanted with or as part of a renal organoid. A renal organoid is a kidney-like structure having a higher-order structure, which has a dendritic branching structure of a collecting duct that connects differentiated nephrons to each other together with differentiated nephrons, nephron precursor cells, and stromal cells consisting of glomeruli and renal tubules. It is an organization.

The renal and glomerulonephropathy is not limited to this, and for example, pathological conditions associated with podocyte disorder, nephrotic syndrome, nephrosclerosis, diabetic nephropathy, chronic glomerulonephritis, or a combination thereof, or diseases thereof. The complications of the disease can be raised, and the condition is preferably accompanied by podocyte disorder.

Hereinafter, the present invention will be specifically described 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 donated by Dr. Satoshi Takahashi of the University of Tsukuba and Dr. Andrew McMahon of the University of Southern California. Mice were fed a CE-2 radiosterilized diet with a 12-hour photocycle and kept in plastic cages. All analyzes were performed with a minimum of 3 litters. In the embryonic stage analysis, noon on the day when the vaginal plug was confirmed in the mated female was regarded as 0.5 days of embryonic stage (E). All animal experiments were conducted according to a protocol approved by the Animal Experiment Committee of Kumamoto University School of Medicine.

Derivation of podocytes from mouse nephron progenitor cells (NPCs)
Hind kidneys were isolated from E15.5 MafB-GFP embryos, finely chopped in PBS using forceps, and incubated in 0.25% trypsin-EDTA for 8 minutes at 37 ° C. to dissociate. The cells were then subjected to the primary antibody staining with anti Robo2 antibody and anti-PDGFR [beta] antibody on ice, and then after the secondary antibody staining, using ^{FACS Robo2 -high / Pdgfrβ - / Mafb} -GFP - sorted population did. For Six2-GFP-Cre embryos, the NPCs, Six2-GFP ⁺ cells or Robo2 ^-high / Pdgfrβ ^- / Podocalyxin ^- were selected as cells. On a 96-well low cell adhesion U-bottom plate, 100,000 NPCs were seeded in a differentiation medium (150 μL) supplemented with a predetermined concentration of CHIR99021 and Y27632 (10 μM). The cells were centrifuged to the bottom and then _{cultured in a 5% CO 2} incubator at 37 ° C. After initial induction with CHIR99021, 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). Medium was changed every 2 days and on day 6 the induced tissue was 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 cultures 201B7 hiPS cell lines from healthy female donors were maintained on iMatrix-511 silk in StemFit AK02N medium. Cells were cultured at 37 ° C. at 5% CO ₂ and passaged every 6-7 days. Medium was changed at 2, 4, 5, and / or 6 days. The NPHS1-GFP knock-in hiPS cell line was prepared according to previous reports (Shamin et al. J. Am. Soc. Nephrol. 27, 1778-1791, 2016) and maintained similarly. The human immortalized podocyte cell line (cell line) was purchased from the University of Bristol. Cells were grown in RPMI medium supplemented with 10% FBS at 33 ° C. with 5% CO ₂ and passaged in 4-5 days. Induction to the podocyte phenotype was performed by culturing the cells at 37 ° C. and 5% CO _{2 for 14 days.}

Induction of podocytes from nephron progenitor cells (NPCs) derived from human iPS cells (hiPSC) According to the report by Oguchi et al. (Non-Patent Documents 6 and 9), spheroids containing NPCs were induced from hiPSC. The induced spheroids were incubated with Accumax at 37 ° C. for 8 minutes to dissociate into single cells. The dissociated cells were stained with anti-ITGA-8 antibody and anti-PDGFRa antibody for 30 minutes on ice for primary antibody staining, followed by secondary antibody staining, and then NPCs were selected as an ^{ITG8 +} / PDGFRa ^{-population using FACS.} On 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). The cells were centrifuged to the bottom and then _{cultured in a 5% CO 2} incubator at 37 ° C. After 24 hours, aggregated cells were transferred onto transwell inserts filled with serum-free differentiation medium supplemented with Fgf9 (10 μg / mL) and IWR-1 (2 μM), SB431542 (5 μM) and retinoic acid (10 μM). The cells were cultured in a medium supplemented with STEP 2 factor (the above step B factor). After 48 hours of culturing, the differentiated cells were transferred to a medium supplemented with STEP 3 factor (the above step C factor) 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 treated with 0.25% trypsin-EDTA for 8 minutes to dissociate. It was blocked with normal mouse serum and _{stained in 1 × HBSS buffer containing 1% BSA and 0.035% LVDS 3} for 30 minutes, followed by staining with a secondary antibody for 10 minutes. Stained cells were analyzed using FACS. Information on the antibodies used is shown in the table below.

Collection of human adult podsites Adult humans from normal sites of renal tissue removed from 3 renal cancer patients after obtaining informed consent in accordance with the approval of the Institutional Review Board of the Graduate School of Life Sciences, Kumamoto University Kidney tissue was obtained. Patients are 49-year-old female, 69-year-old male and 80-year-old male. All patients showed normal renal function and normal urinary findings. The normal site of renal tissue removed from the patient was 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) and then digested with 0.25% trypsin-EDTA. After cell dissociation, erythrocytes were destroyed using RBC lysis buffer. Thereafter, the anti-human Nephrin antibody, and subjected to primary antibody staining with anti-human Podocalyxin antibody, then after the secondary antibody staining, using FACS, normal human podocyte was isolated as Nephrin ⁺ / Podocalyxin ⁺ population.

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

Immunohistochemical analysis Samples were fixed with 10% formalin and embedded in paraffin to obtain 6 mm thick sections. Antigen recovery was performed with a citrate buffer solution having a pH of 6.0. Sections were blocked with PBS containing 1% BSA and incubated with primary antibody overnight at 4 ° C. Staining with a secondary antibody conjugated to Alexa488, 568, 594, 633 or 647. Nuclei were stained with DAPI. A fluorescence image was taken with a confocal microscope. The antibody information used is shown below.

Electron microscope imaging Observation was performed with an immunoelectron microscope. As an antibody, an antibody against the N-terminal domain of Nephrin was used. The prepared sections were incubated overnight with the antibody and then with a secondary antibody bound to 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
In order to analyze the signals required for the differentiation of NPCs into podocytes, we established a culture system capable of detecting the differentiation of NPCs purified from mouse embryo kidney into nephron epithelium and podocytes. Previous reports have shown that NPCs express Robo2 and Pdgfrb is negative. Therefore, flow cytometric analysis was performed using Six2-GFP transgenic mice, and it was confirmed that the ^Robo2-high / Pdgfrb ^- fraction of the kidney of E15.5 was Six2-GFP ^{+ NPCs.} In addition, to eliminate podocytes present in ^{the Robo2-high} / Pdgfrb ^- fraction, we have established a method for isolating Six2-GFP + NPCs with high purity, excluding ^{the Podocalyxin + fraction.} In order to quantitatively evaluate podocyte induction, MafB-GFP knock-in mice (MafB-GFP mice) showing GFP expression only in differentiated podocytes were used. ^{Robo2 -high / Pdgfrb - / MafB-} GFP - cell fraction, as expected, Six2, Pax2, Sall, and is expressed the NPC-specific genes, such as Cited1 in Six2-GFP ⁺ cells the same level, the NPCs It was confirmed that it could be isolated with high purity. The results are shown in FIG.

Example 2: Examination of Wnt Signals Essential for Differentiation into METs and 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, the effect of Wnt signal intensity and duration on podocyte differentiation was unclear.
Therefore, we first examined the concentration and duration of Wnt signaling required to induce mesenchymal epithelial transition from NPCs. Using the NPCs prepared in Example 1, CHIR99021 (CHIR) was used as a Wnt agonist, treated at a concentration of 1 to 10 μM for 12 to 48 hours, and analyzed on the 6th day. The outline is shown in FIG. After initial CHIR treatment, 0.5 μM CHIR was continuously added as basal medium to maintain tissue growth and survival. Treatment of NPCs with a combination of 3-5 μM CHIR and 24-48 hours was found to result 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 FIG. 5, 24-hour CHIR treatment of 3 μM significantly induced podocytes of Mafb-GFP +, whereas treatment of 5 μM and 48 hours hardly induced podocytes. rice field. From this, it was found that when NPCs derived from mouse embryo kidneys were used, the 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 in the epithelialization of NPCs, and that early Wnt signaling has a significant effect on the final podocyte formation. ing.

Example 3: Inhibition of TGF-β signaling in renal epithelial precursor aggregate (PA) to renal corpuscle (RV) differentiation
To study the process of inducing NPCs to podocytes, we defined an intermediate process in the in vitro culture system described above. The result of qRT-PCR is shown in FIG. From this, it was clarified that it corresponds to the differentiation stage of PA at 24 hours after the start of induction and RV at 48 hours.

Direct differentiation from NPCs to proximal RVs to podocytes, as RV has already polarized along the proximal distal axis and the proximal region of RV is presumed to contain podocyte precursors. Based on the hypothesis that the induction efficiency of podocytes is increased, the following studies were conducted.
The effect of the growth factor or inhibitor in the PA to RV differentiation step (24 hours to 48 hours: STEP 2) was confirmed. High concentrations of CHIR (2 μM) suppressed the final percentage of podocytes compared to the reference condition of 0.5 μM CHIR. In addition, IWR-1, a Wnt signaling inhibitor, inhibited epithelialization. In contrast, the Tgf-β signaling pathway inhibitor SB431542 (SB) significantly increased the proportion of final podocytes. As a result of similar examination of other factors, activin A, retinoic acid (RA), and RA antagonist BMS493 did not increase the efficiency of podocyte induction. The results are shown in FIGS. 7 and 8.

When the gene expression profile at the RV stage (48 hours) was examined, the expression of the proximal region markers Wt1, MafB, and Foxc2 was enhanced by the treatment of SB (Fig. 9). On the other hand, the distal marker of RV was slightly reduced compared to the reference condition (0.5 μM CHIR). In addition, SB treatment increased both the ratio and number of ^{Wt1 + proximal RV cells.} On the other hand, high concentrations of 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 differentiation into proximal RVs, resulting in an increase in the proportion and number of final podocytes. ..

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

In the examination of the cell number on the 6th day, the SB treatment did not significantly affect the final podocyte number, while the cell number of LTL-positive proximal tubular cells decreased. The number of cells in the Cdh1-positive distal tubule tended to decrease, but the difference was not significant. The results are shown in FIG. Furthermore, as a result of gene expression analysis, the expression of podocyte-related genes was increased, while the expression of genes related to the nephron fraction other than podocytes was decreased. The results are shown in FIG.
The podocytes prepared using the high-efficiency induction method of the present invention were not only positive for MafB-GFP, but also expressed a series of typical podocyte-specific proteins (Fig. 13, bottom).
These results show that inhibition of TGF-β signaling at the stage of differentiation into each nephron site (STEP 3) promotes differentiation into podocytes and suppresses differentiation into other nephron sites, thereby inducing podocytes. It suggests increasing efficiency.

Example 5: Induction of selective podocytes from human iPS cells Next, efficient induction to podocytes was examined using nephron progenitor cells induced from human iPS cells. The Nephrin-GFP hiPS cell line was used to monitor the efficiency of induction from hiPSC to podocytes. A three-step podocyte differentiation induction method established in mouse NPCs was applied to hiPS. The outline is shown in FIG. First, the concentration and duration of Wnt agonists in the NPCs to PA stage (STEP 1) were examined, and then the culture was carried out under the reference conditions (0.5 μM CHIR99021) until the 9th day. The induction efficiency of podocytes was confirmed by the ^{Nephrin-GFP + cell ratio.} The results are shown in FIG. 24-hour treatment with 3 μM CHIR99021 was found to be the most efficient for podocyte induction.

Results of histological analysis, the CHIR induction for 24 hours, NPCs ^{may, PA (LHX1 + / ECAD -} ) differentiate into, it was confirmed that the form RV (LHX1 ⁺ / ECAD ⁺⁾ at 48 hours.
In the process of PA to RV differentiation, SB431542 treatment increased the proportion of podocytes, as in mice. In addition, in the case of hiPSC-derived NPCs, the addition of Wnt signaling inhibitors (IWR1) and RA in STEP 2 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, administration of SB, IWR1 and RA alone was effective in increasing the efficiency of podocyte induction. Furthermore, by combining SB and IWR1, the induction efficiency of podocytes was further increased, reaching 90% or more (Fig. 17).
As described above, the selective induction of podocytes from hiPSCs-derived NPCs was achieved by examining the necessary culture conditions for each STEP and optimizing them. The outline of the process is shown in FIG. 1b. It was also clarified that this induction method can be applied to the hiPS cell line (201B7) without gene editing. The yield of podocytes themselves was also about three times that of normal renal organoids, as 300,000 podocytes could be induced from 10,000 hiPSCs (Fig. 18). In addition, STEP 3 was able to continue culturing even after the 9th day after the start of culturing, and the cells were still alive 15 days after the start of culturing.

Example 6: Comparison of gene expression profiles of induced podocytes, human podocytes, and cell lines The gene expression profiles of induced podocytes were compared between human adult kidney-derived podocytes and human immortalized podocyte cell lines. As a result of the analysis, the induced podocytes showed high expression levels of podocyte-specific genes such as NPHS1, NPHS2, WT1, and SYNPO, and these expression levels were comparable to those of human adult podocytes (Fig. 19). .. In the cell line, the expression of SYNPO was relatively maintained as compared with the adult human podocytes and the 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 FIG. 20, it was confirmed that WT1 was expressed in the nucleus and NEPHERIN was expressed on the cell boundary in the induced podocytes. .. Other slit membrane-related proteins, NEPH1 and PODOCIN, were also localized along with NEPHERIN. As a result of transmission electron microscopic analysis, the presence of podocytes on the basal side of podocytes was also confirmed (Fig. 21). By staining with an anti-nephrin antibody, it was also confirmed that these foot processes express nephrin protein (Fig. 22). A slit film-like structure was also confirmed as a filamentous crosslink 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, the inducible podocytes expressed a large amount of slit membrane-related proteins such as NEPHRIN, phosphorylated NEPHRIN (p-NEPHRIN), NEPH1, and PODOCIN. Inducible podocytes also expressed the differentiated podocyte marker SYNAPTOPODIN, and the endogenous antigen of membranous nephropathy, PLA2R, which were higher than in hiPSC and cell lines. These results suggest that the inducible podocytes express a sufficient amount of protein associated with the podocytes.

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

Podocyte disorder is the most important pathological condition as a cause of urinary protein, and the development of a drug that acts protectively against podocytes is awaited in kidney diseases such as diabetic nephropathy and chronic glomerulonephritis. The podocytes produced by the induction method of the present invention express a large amount of podocyte-related proteins, and screening for the development of a drug that acts protectively on podocytes in the event of a disorder, that is, in the area of kidney disease. It is considered to be useful in drug discovery. Inducible podocytes can also be applied as a material for cytotoxicity tests when verifying the toxicity of drugs that may cause renal damage.
Furthermore, by inducing patient-derived podocytes from iPS cells obtained from patients with congenital nephrotic syndrome, which is a disease related to podocytes, it can be applied to elucidation of the pathophysiology of rare diseases and development of therapeutic methods. In addition, since it has become possible to edit genes in iPS cells using technologies such as CRISPR / CAS9 and TALEN, we have established a cell line in which a specific gene has been removed or overexpressed. By applying the induction method of the present invention, it is possible to apply it to an experimental system for examining what role the gene plays in human podocytes.

The above detailed description merely describes the object and object of the present invention, and does not limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein, without departing from the appended claims.

According to the induction method of the present invention, podocytes can be efficiently induced. In addition, the podocytes derived by the method of the present invention express many podocyte-related proteins and are useful as mimics of podocytes.

Claims (26)

A method for inducing differentiation of podocytes, the following steps:
Step A: A step of culturing a nephron progenitor cell or a population thereof in a medium containing a Wnt agonist and a ROCK inhibitor.
Step B: A step of culturing the cells or a population thereof obtained in Step A in a medium containing Fgf, and
Step C: A step of culturing the cells or a population thereof obtained in Step B in a medium containing Fgf.
How to include
Here, at least one of the media in step B or step C contains a TGFβ signaling pathway inhibitor. The method according to claim 1, wherein the concentration of the Wnt agonist in the medium used in step A is more than 1 μM and less than 10 μM. The method according to claim 1 or 2, wherein the medium in steps B and C further comprises a Wnt agonist having a concentration of about 1 μM or less. The method according to any one of claims 1 to 3, wherein the culture in the step A is 12 hours to 48 hours, and the culture in the step B is 12 hours to 48 hours. The method according to claim 4, wherein the culture in the step C is 2 to 30 days. The method according to any one of claims 1 to 5, wherein the Wnt agonist is CHIR99021 and the TGFβ signaling pathway inhibitor is SB431542. The method according to any one of claims 1 to 6, wherein the nephron progenitor cell is a nephron progenitor cell derived from a mammalian-derived iPS cell. The method according to claim 7, wherein the iPS cells are human-derived iPS cells. The method of claim 7 or 8, wherein the medium in step B and / or step C further comprises a Wnt signal inhibitor. The method according to any one of claims 7 to 9, wherein the medium in step B and / or step C further comprises retinoic acid (RA) or an RA analog. The nephron progenitor cells are subjected to the following steps:
(A) A step of culturing an embryoid body derived from human iPS cells in a medium containing a high concentration (concentration A) Wnt agonist.
(B) The step of culturing the embryoid body in a medium containing Bmp, actibine, retinoic acid, and a medium concentration (concentration B) of Wnt agonist, and (c) the embryoid body being Fgf and low. The step of culturing in a medium containing a Wnt agonist of a concentration (concentration C),
(Here, the concentration of the Wnt agonist is concentration A> concentration B> concentration C, and the concentration A is at least 5 times the concentration C).
The method according to any one of claims 7 to 10, which is derived from human iPS cells. The Wnt agonist in the steps (a), (b) and (c) is CHIR99021, and the concentrations A and C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively. The Bmp in the 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 the step (b) is 2.5 to 40 ng / mL. The method of claim 11, which is the concentration. A medium kit for inducing differentiation of podocytes, which comprises a differentiation-inducing medium and three containers containing each of the components described below.
(1) A first container containing a Wnt agonist and a ROCK inhibitor,
(2) A second vessel containing TGFβ signaling pathway inhibitor, Wnt signaling inhibitor, retinoic acid (RA) or RA analog, and Fgf, and (3) TGFβ signaling pathway inhibitor, Wnt signal inhibitor, retinoic acid ( RA) or RA analog, and a third container containing Fgf. The 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, the following steps:
(I) A step of inducing differentiation of nephron progenitor cells or a population thereof from a mammalian-derived iPS cell or a population thereof.
(Ii) A step of 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) A step of culturing the cells or a population thereof obtained in step (ii) in a medium containing Fgf, and
(Iv) A step of culturing the cells or a population thereof obtained in step (iii) in a medium containing Fgf.
How to include
Here, at least one of the media in step (iii) or step (iv) contains a TGFβ signal pathway inhibitor, and at least one of the media in step (iii) or step (iv) contains a Wnt signal inhibitor, and At least one of the media in step (iii) or step (iv) comprises retinoic acid (RA) or RA analog. The method according to claim 15, wherein the concentration of the Wnt agonist in the medium used in the step (ii) is more than 1 μM and less than 10 μM. The method of claim 15 or 16, wherein the medium in steps (iii) and (iv) further comprises a Wnt agonist at a concentration of about 1 μM or less. The method according to any one of claims 15 to 17, wherein the iPS cell is a human-derived iPS cell. The method according to any one of claims 15 to 18, wherein the culture in the step (ii) is 12 hours to 48 hours, and the culture in the step (iii) is 12 hours to 48 hours. The method according to claim 17, wherein the culture in the step (iv) is 2 to 30 days. The method according to any one of claims 15 to 18, wherein the Wnt agonist is CHIR99021, the TGFβ signaling pathway inhibitor is SB431542, and the Wnt signal inhibitor is IWR-1. The step (i) is the following step:
(A) A step of culturing an embryoid body derived from human iPS cells in a medium containing a high concentration (concentration A) Wnt agonist.
(B) The step of culturing the embryoid body in a medium containing Bmp, actibine, retinoic acid, and a medium concentration (concentration B) of Wnt agonist, and (c) the embryoid body being Fgf and low. The step of culturing in a medium containing a Wnt agonist of a concentration (concentration C),
(Here, the concentration of the Wnt agonist is concentration A> concentration B> concentration C, and the concentration A is at least 5 times the concentration C).
The method according to any one of claims 15 to 21. The Wnt agonist in the steps (a), (b) and (c) is CHIR99021, and the concentrations A and C are 7.5 μM to 15 μM and 0.5 μM to 2.0 μM, respectively. The Bmp in the 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 the step (b) is 2.5 ng / mL to 40 ng / ml. The method of claim 20, wherein the concentration is mL. A method for evaluating the effect or effect of a test substance on podocytes, wherein a cell population of podocytes prepared by the method according to any one of claims 1 to 12, 15 to 23 in the presence of the test substance is used. From the group consisting of the following items related to the cells: cell proliferation, cell survival, cell damage, protein expression level, gene expression level, albumin uptake ability, albumin filtration ability, autophagy activity and intracellular transmission signal. A method comprising measuring at least one item selected and assessing the effect of the test substance on podocytes. A method for screening a substance for treating or ameliorating a podocyte disorder, wherein a podocyte cell population prepared by the method according to any one of (x) claims 1 to 12 and 15 to 23 is subjected to podocyte disorder. At least one item selected from the group consisting of the step of culturing in the presence of the indicated drug and test substance, and (y) survival, proliferation, alleviation of damage, change in protein expression level, and change in gene expression level of podocyte cells. How to screen for candidate substances to treat or ameliorate podocyte disorders, including the step of measuring
Here, the culture of the podocyte cell population in step (x) may be any of the following:
Cultivate the podocyte cell population in the presence of (x-1) podocyte-damaging drug and test substance at the same time, or cultivate the podocyte cell population in the presence of (x-1) podocyte-damaging drug. Then, the damaged podocyte cell population is cultured in the presence of the test substance. The screening method according to claim 25, wherein the drug exhibiting podocyte damage is at least one drug selected from the group consisting of puromycin aminonucleoside, doxorubicin, angiotensin II, and an anti-podocyte antibody.

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