WO2015056756A1 - 多能性幹細胞からの腎臓誘導法 - Google Patents
多能性幹細胞からの腎臓誘導法 Download PDFInfo
- Publication number
- WO2015056756A1 WO2015056756A1 PCT/JP2014/077601 JP2014077601W WO2015056756A1 WO 2015056756 A1 WO2015056756 A1 WO 2015056756A1 JP 2014077601 W JP2014077601 W JP 2014077601W WO 2015056756 A1 WO2015056756 A1 WO 2015056756A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- concentration
- cells
- cell
- medium
- differentiation
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
- C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
- C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Definitions
- the present invention relates to a method for inducing kidney from pluripotent stem cells. More specifically, the present invention relates to a method for deriving a three-dimensional structure of the kidney including both glomeruli and tubules from pluripotent stem cells such as ES cells and iPS cells.
- the kidneys play an important role in maintaining the homeostasis of electrolytes and water in the body at the same time as discharging waste products by producing urine.
- renal function When renal function is lost, water and various toxic components accumulate, leading to consciousness opacification, dyspnea due to pulmonary edema, and death due to hyperkalemia.
- the number of patients receiving artificial dialysis in Japan has reached about 300,000, and is still increasing. Diabetes is the first cause of introducing dialysis, accounting for about 45% of the number of patients introduced.
- the kidney plays an important role as an endocrine organ, regulates blood pressure by producing renin, and is involved in bone metabolism and erythrocyte maintenance by activating vitamin D and producing erythropoietin.
- erythropoietin is administered several times a week for anemia associated with renal failure, but it is necessary to administer it throughout the life of the patient, resulting in an increase in medical costs.
- kidney transplantation is a fundamental treatment that can completely compensate for impaired renal function, but it cannot be a common treatment due to chronic donor shortages.
- Artificial dialysis imposes strict dietary restrictions and regular visits to the patient, but it only pays for the filtering function of the kidneys and causes long-term complications. Regenerative therapy is attracting attention as a new alternative treatment.
- kidneys in vitro In spite of the successful construction of various types of tissues from pluripotent stem cells in vitro, the creation of kidneys in vitro remains unsuccessful, and the establishment of a methodology is awaited. This is mainly due to the lack of elucidation of the mechanism due to the developmental complexity of the renal differentiation process in vivo.
- the kidney is different from other major organs, and in the process of development, after two temporary primordiums (anterior and intermediate kidneys) located in the front and further located in the rear and differentiated into adult kidneys It is formed through a complex process involving the formation of the three primordia of the kidney.
- it is essential to construct a three-dimensional structure that includes both the glomeruli and tubules that make up the functional unit “nephron”. Something further increases the technical difficulty.
- the kidney develops from the metanephros that develops most backward in the fetal trunk.
- the metanephros are formed by the interaction between two progenitor tissues, the metanephric mesenchyme and the ureteric bud. So far, cell lineage analysis has shown that both metanephric mesenchyme and ureteric buds originate from an intermediate mesoderm expressing the transcription factor Osr1, which appears around embryonic day (E) 8.5.
- Patent Literature 1 Mugford et al., Dev Biol 324, 88-98, 2008). However, the underlying mechanism for what growth factor signals the early mesoderm differentiates into the intermediate mesoderm has not been clarified.
- Non-Patent Document 2 Mugford et al., Dev Biol 319, 396-405, 2008; Non-Patent Document 3: Wellik et al., Genes Dev 16, 1423-1432, 2002).
- the posterior Hox gene is expressed, and the metanephric mesenchyme is formed (posteriorization) has not yet been elucidated.
- the kidney is formed by the interaction of two tissues, the mesodermal-derived metanephric mesenchyme and the ureteric bud, but the main components such as “glomerulus” and “tubule” contained in the functional unit nephron.
- the structure is derived from the former (posterior renal mesenchyme).
- progenitor cells nephron nephron progenitor cells
- Non-patent Document 4 Osafune, Nishinakamura et al., Development 133, 151-161, 2006).
- Patent Document 1 a method for inducing intermediate mesoderm from iPS cells by culturing iPS cells in the presence of activin A and Wnt and then in the presence of BMP and Wnt has been reported.
- Patent Document 1 a method for inducing intermediate mesoderm from iPS cells by culturing iPS cells in the presence of activin A and Wnt and then in the presence of BMP and Wnt has been reported (Patent Document 1).
- Patent Document 1 a method for inducing intermediate mesoderm from iPS cells by culturing iPS cells in the presence of activin A and Wnt and then in the presence of BMP and Wnt has been reported.
- Patent Document 1 a method for inducing intermediate mesoderm from iPS cells by culturing iPS cells in the presence of activin A and Wnt and then in the presence of BMP and Wnt has been reported (Patent Document 1).
- An object of the present invention is to provide a process or method that can be used for inducing a three-dimensional structure of a kidney from pluripotent stem cells such as ES cells or iPS cells.
- the present invention first induces intermediate mesoderm cells from ES cells and iPS cells (for example, mouse embryonic stem cells and human induced pluripotent stem (iPS) cells), and further, from intermediate mesoderm cells to metanephros
- the object is to provide a method for inducing lobe cells (post-renal nephron progenitor cells), and a new model of kidney differentiation line using such a method.
- the present inventors have found that the three-dimensional structure of the kidney, that is, the tubule and glomerulus, can be reconstructed from mouse and human pluripotent stem cells.
- the present invention was completed by successfully inducing nephron progenitor cells (post-renal mesenchyme).
- nephron progenitor cells post-renal mesenchyme.
- Most epithelial cells constituting the kidney are derived from metanephric mesenchymal cells contained in the metanephros located at the posterior end of the trunk.
- it has been difficult to attempt in vitro renal regeneration because it undergoes a complicated development process including the formation of the pronephros and middle kidneys prior to the development of the metanephros.
- metanephric mesenchymal cells which are three-dimensional kidneys that include glomeruli with glomerular epithelial cells and tubules with distinct lumens.
- the present invention has been completed by finding that the structure can be reconstructed.
- the present invention has made it possible to reconstruct the kidney in vitro.
- the present invention includes the following.
- a method for inducing differentiation of a metanephric mesenchyme, which is a (nephron) nephron progenitor cell, from a pluripotent stem cell derived from a mammal comprising the following three steps: (A) culturing embryoid bodies derived from pluripotent stem cells in a medium containing Bmp and a high concentration (concentration A) of a Wnt agonist; (B) culturing the embryoid body in a medium containing Bmp and a medium concentration (concentration B) Wnt agonist; and (c) the embryoid body is Fgf and a low concentration (concentration C) Wnt agonist.
- the Wnt agonist is selected from the group consisting of CHIR99021, BIO, and SB415286 (however, the Wnt agonist in each step may be the same or different). The differentiation induction method described.
- the Bmp is selected from the group consisting of the Bmp family, preferably from the group consisting of Bmp2, Bmp4 and Bmp7
- the Fgf is from the group consisting of the Fgf family, preferably from the group consisting of Fgf2, Fgf9 and Fgf20
- the Wnt agonist in the steps (a), (b) and (c) is CHIR99021, the concentration A is 7.5 ⁇ M to 15 ⁇ M, and the concentration C is 0.5 ⁇ M to 2.0 ⁇ M.
- the medium is a medium containing neither Bmp, retinoic acid or activin.
- the pluripotent stem cells are mouse ES cells or iPS cells, and the step (a) is a step of culturing at least 1 day or more and 4 days or less (preferably, at least once, the medium is replaced with a fresh medium.
- the step in which the pluripotent stem cell is a human ES cell or iPS cell and the step (a) is cultured for at least 3 days to 11 days (preferably, at least twice, the medium is replaced with a fresh medium)
- a nephron progenitor cell induced to differentiate from a pluripotent stem cell derived from a mammal which is a cell population that expresses all of transcription factors Osr1, Wt1, Pax2, Six2, Hoxa10, and Hoxa11.
- (21) A method for producing a three-dimensional kidney structure having glomeruli and tubules using the nephron progenitor cells according to any one of (18) to (20).
- (24) A proximal tubule cell, characterized in that it is a cell population expressing Cadherin6, Megalin, and LTL induced to differentiate from the nephron progenitor cell according to any one of (18) to (20). .
- (25) The proximal tubule cell according to (24), wherein the differentiation induction is performed by co-culturing nephron progenitor cells with fetal spinal cord or Wnt4-expressing cells.
- a distal cell which is a cell population expressing E-cadherin, Brn1, and NCC, which is induced to differentiate from the nephron progenitor cell according to any one of (18) to (20).
- Tubule cells The distal tubule cell according to (26), wherein the differentiation induction is performed by co-culturing nephron progenitor cells with fetal spinal cord or Wnt4-expressing cells.
- a glomerular epithelial cell characterized by being a cell population expressing Wt1, Nephrin, and Podocin, which is induced to differentiate from the nephron progenitor cell according to any one of (18) to (20). .
- a differentiation-inducing medium kit for inducing nephron progenitor cells from pluripotent stem cells comprising (i) a differentiation-inducing medium, and (ii) a Wnt agonist, At least three differentiation-inducing media containing agonist in stepwise concentrations are prepared, wherein the stepwise concentration comprises at least concentrations A, B and C, where concentration A> concentration B> concentration C, A is at least 5 times the concentration C, A culture medium kit and a culture medium kit.
- the stepwise concentration of the Wnt agonist is such that the differentiation-inducing medium is adjusted so that the concentration A is at least 3 times the concentration B and the concentration B is at least 3 times the concentration C.
- metanephric nephron progenitor cells can be efficiently induced from stem cells (for example, ES cells and iPS cells), and further, reconstruction of the three-dimensional structure of the kidney including both glomeruli and tubules can be achieved. It has become possible. This opened up the possibility of creating disease-specific tubules and glomerular epithelial cells using iPS cells established from patients with various diseases, and applying them to elucidating their pathologies and developing new drugs. . Furthermore, since these metanephric nephron progenitor cells formed glomeruli with vascular invasion by transplanting into immunodeficient mice, it is expected to lead to the regeneration of functional kidneys capable of producing urine in the future. Is done.
- stem cells for example, ES cells and iPS cells
- mouth ES cell is shown.
- A represents activin
- B represents Bmp4,
- C represents CHIR99021, R represents retinoic acid, and
- F represents Fgf9.
- C10, C3, and C1 represent 10 ⁇ M, 3 ⁇ M, and 1 ⁇ M CHIR99021, respectively.
- derivation of a metanephric nephron progenitor cell (metanephric mesenchyme) from a human iPS cell is shown.
- FIG. 2 shows a target strategy for creating an Osr1-GFP knock-in mouse.
- EGFP was inserted into the Osr1 locus so that the N-terminal amino acid of Osr1 was bound to EGFP.
- B represents BamHI. Shows EGFP expression at E8.5 (A), E9.5 (B) and E15.5 (C) in Osr1-GFP knock-in mice.
- the scale bar is 500 ⁇ m.
- the bright field image (left), Pax2 expression (middle), and LTL expression (right) of the colonies formed by the Osr1-GFP positive population are shown.
- the scale bar is 100 ⁇ m.
- kidney-related genes in colonies formed by Osr1-GFP positive population is shown relative to ⁇ -actin. Each first lane shows only Wnt4 feeder cells without colonies.
- the markers are kidney lineage markers: Pax2 and Sall1; proximal tubule markers: Slc5a1; Henleloop markers: Clcnka and Clcnkkb; distal tubule markers: Pou3f3; connecting tubule markers: s100g.
- Itga8 and Pdgfra in E8.5, E9.5, E11.5, and E15.5 of Osr1-GFP knock-in mice is shown by immunostaining of embryo sections.
- FIG. 7 shows the results of FACS analysis of Itga8 / Pdgfra expression in a Sal1-GFP high population (left) and a Six2-GFP positive population (right) in E11.5 metanephros of Osr1-GFP knock-in mice.
- the anterior part (somatic level 7-22) and posterior part (posterior from somite level 23) of the embryo were manually isolated and 20,000 cells of Osr1 + cells were seeded on Wnt signal feeder cells. The colony formation rate of each part is shown on the right side as mean ⁇ standard error (n 3). Various markers of nephron progenitor cells in E9.5 intermediate mesoderm were confirmed by immunostaining. The intermediate mesoderm is outlined with a dashed line. The scale bar is 50 ⁇ m. Six2-GFPCreER mice were bred with mice bearing the tdTomato reporter gene and tamoxifen was injected at E8.75 to temporarily activate Cre around E9.5.
- the metanephric nephron progenitor cell marker genes such as Osr1, Wt1, Pax2, Six2, Gdnf and Crym were expressed in both E9.5 and E11.5 Osr1 + / Itga8 + / Pdgfra ⁇ populations.
- tail Hox genes such as Hoxa10, a11, c10, d10, d11 and d12 were concentrated in the E11.5 metanephric mesenchyme. It is a schematic diagram of mouse kidney development. The E8.5 T + / Cdx2 + / Tbx6 + posterior undifferentiated mesoderm produces the metanephric mesenchyme via the posterior intermediate mesoderm.
- the intermediate mesoderm located behind E9.5 that does not express Pax2 and Six2 or form colonies expresses the tail Hox gene.
- the metanephric mesenchyme located behind E10.5 expresses the tail Hox gene, Pax2 and Six2, and forms many colonies on Wnt4 feeder cells.
- Y Y27632 (Rock inhibitor); SU: SU5402 (Fgfr1 inhibitor); C1: 1 ⁇ M CHIR99021 (canonical Wnt agonist); C3: 3 ⁇ M CHIR99021, C10: 10 ⁇ M CHIR99021; RA: retinoic acid.
- Y Y27632 (Rock inhibitor); F: Fgf9; C1: 1 ⁇ M CHIR99021.
- a lineage segregation model of ureteric buds from the mesoderm and metanephric mesenchyme is shown. It is the result of the induction
- C and D showed culture conditions.
- E10.5MM indicates the E10.5 metanephric mesenchyme, and T-GFP + indicates the sample at the start of induction.
- CHIR CHIR99021 (canonical Wnt agonist); A10: 10 ng / ml activin; SB: SB431542 (Smad2 / 3 inhibitor); LDN: LDN93189 (Smad1 / 5/8 antagonist); XAV: XAV939 (Wnt signal antagonist), R : 0.1 ⁇ M retinoic acid; BMS: BMS493 (pan-RAR antagonist), F: 5 ng / ml Fgf9; SU: SU5402 (Fgfr1 inhibitor). It is the result which confirmed the expression of the marker gene in the induction
- A is the result of confirming the dynamics of the temporal expression of the marker gene.
- B is the result of immunostaining of induced cell aggregates. High magnification images are shown in the right two panels. The scale bar is 200 ⁇ m (6 panels on the left) and 20 microns (2 panels on the right end).
- C is the result of FACS analysis of Itga8 / Pdgfra expression on day 4 of induction (day 4). The kinetics of gene expression at each stage of induction of nephron progenitor cells from mouse ES cells is shown.
- FIG. 6 shows the localization of Osr1-GFP / Wt1 / Pax2 and Osr1-GFP / Sall1 / Six2 positive cells in the induced embryoid body at day 8.5 in the induction of metanephric nephron progenitor cells from mouse ES cells.
- the scale bar is 200 ⁇ m (6 panels on the left).
- FIG. 6 shows the results of FACS analysis of Osr1-GFP and Itga8 / Pdgfra expression in embryoid bodies at day 8.5 in the induction of nephron progenitor cells from mouse ES cells.
- mouth ES cell is shown.
- AD are the results of hematoxylin and eosin staining (B and D: high magnification).
- a and B are embryonic retrorenal mesenchyme, and C and D are induced embryoid bodies co-cultured with spinal cord. Kidney tubules are outlined by dashed lines. SP: spinal cord, G: glomerulus.
- the scale bars are 200 ⁇ m and 20 ⁇ m.
- E to P show induced embryoid bodies as tubule markers (E to H and L to P) (proximal tubule markers: LTL, distal tubule markers: E-cadherin, Brn1 and NCC) and glomeruli. The result is immunostaining with markers (I to K).
- the scale bars are 100 ⁇ m (E, I) and 20 ⁇ m (FH, JL).
- mouth ES cell is shown.
- Q and R show the kidneys of mice that had been induced embryonic bodies transplanted under the kidney capsule of nude mice and removed one week later.
- the graft is outlined with a dashed line.
- the black arrow in Q indicates graft angiogenesis.
- R is a fluorescent image of the graft.
- the white arrow on R indicates a nephron-like structure in the graft.
- the scale bar is 500 ⁇ m.
- S to U are the results of hematoxylin and eosin staining of transplanted kidney sections.
- S is a low-magnification image (g: graft; k: host kidney, scale bar: 100 ⁇ m).
- T is a medium magnification image (the renal tubule is outlined by a broken line, G: glomerulus, scale bar: 20 ⁇ m).
- U is a high-magnification image of the glomerulus in the graft (arrows indicate red blood cells in the vasculature that enter the glomerulus, scale bar: 20 ⁇ m).
- V and W are the results of immunostaining with Pecam1, a vascular marker (V: low magnification image, W: high magnification image of glomeruli in the graft, g: graft, k: host kidney, scale Bar: 20 ⁇ m).
- X and Y indicate the expression of blood vessel inducing factors (X: Vegfa, Y: Efnb2) in glomerular epithelial cells.
- FIG. 4 shows the localization of Wt1 / Pax2 and Sall1 / Six2 positive cells in induced embryoid bodies at day 14 (day 14) in the induction of metanephric nephron progenitor cells from human iPS cells.
- the scale bar is 75 ⁇ m.
- D and E are the results of hematoxylin and eosin staining of induced embryoid bodies co-cultured with the spinal cord (E is a high magnification image).
- the tubule was outlined with a broken line.
- SP spinal cord
- G glomerulus
- scale bar 200 ⁇ m (D) and 20 ⁇ m (E).
- F to K are the results of immunostaining of adjacent sections of induced embryoid bodies.
- K is a glomerular marker
- H and I are tubular markers
- the scale bar is 50 ⁇ m. It is a differentiation induction model from pluripotent cells to metanephric mesenchyme.
- the inventors succeeded in inducing metanephric nephron progenitor cells capable of reconstructing a three-dimensional kidney including tubules and glomeruli from mouse and human pluripotent stem cells. Since tubules and glomeruli are two important components for kidney function, the method of the present invention and the findings obtained using it reveal the molecular mechanisms underlying human kidney disease. Therefore, it can be used for regenerative medicine.
- the data presented here also demonstrates that kidney development is well conserved between humans and mice, and demonstrates the robustness of the method of the present invention that faithfully reproduces physiological processes. Show.
- T transcription factor Brachyury
- Non-patent document 5 Takemoto et al., Nature 470, 394-398, 2011; Non-patent document 6: Tzouanacou et al., Dev Cell 17, -365-376, ; 2009; Non-patent document 7: Wilson Development Develop136, 1591-1604, 2009).
- this recently identified posterior undifferentiated mesoderm may be the origin of the metanephric mesenchyme, nephron progenitor cells, This counters the conventional idea that is derived from the anterior middle mesoderm.
- the introduction of the somatic stem cell induction model may be applied to induce differentiation of organs located on the other caudal side.
- the present inventors have made cells T-positive by utilizing Wnt agonist at a concentration higher than that normally used for maintaining the undifferentiated state of mouse ES cells.
- the Wnt agonist concentration is gradually decreased in stages, and further differentiation factors are added in combination with the differentiation stage-specific growth factors to induce differentiation into the kidney lineage, eventually allowing the formation of metanephric nephron progenitor cells.
- the need for high concentrations of Wnt agonists in the early stages of induction reflects the importance of Wnt signals for tail trunk elongation and axial stem cell maintenance in vivo.
- the “pluripotent stem cell” used in the present invention refers to a cell having self-replicating ability, capable of being cultured in vitro, and capable of differentiating into cells constituting an individual.
- ES cells embryonic stem cells
- iPS cells induced pluripotent stem cells derived from somatic cells
- iPS cells or ES cells are particularly preferably used in the present invention.
- mouse iPS cells and ES cells are particularly preferably used in the present invention.
- human iPS cells and ES cells are particularly preferred.
- the ES cell used in the present invention is not particularly limited as long as it is an ES cell derived from a mammal.
- mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, cows, horses, goats, monkeys, and humans, with mice and humans being preferred.
- ES cells are cultured by cultivating fertilized eggs at the blastocyst stage together with feeder cells, dissociating the proliferated cells derived from the inner cell mass, and further repeating the transplanting operation. It can be established as a stock.
- iPS cells artificial pluripotent stem cells
- transcription factors differentiation
- fibroblasts e.g, fibroblasts
- a pluripotent factor is a cell that has acquired differentiation pluripotency equivalent to that of an ES cell by introducing a gene. Many factors have been reported as the “pluripotency factor” and are not particularly limited.
- the Oct family eg, Oct3 / 4
- the Sox family eg, Sox2, Sox1, Sox3, Sox15 and Sox17
- Klf family eg, Klf4, Klf2, etc.
- Myc family eg, c-Myc, N-Myc, L-Myc, etc.
- Nanog LIN28 and the like.
- Mammal-derived ES cells can be cultured by a conventional method. For example, using mouse embryonic fibroblasts (MEF cells) as feeder cells, leukemia inhibitory factor (LIF), KSR (knockout serum substitute), fetal bovine serum (FBS), non-essential amino acids, L-glutamine, pyruvate, It can be maintained using a medium supplemented with penicillin, streptomycin, or ⁇ -mercaptoethanol, such as a DMEM medium.
- iPS cells can also be cultured by a conventional method.
- a medium containing bFGF, KSR (knockout serum substitute), non-essential amino acids, L-glutamine, penicillin, streptomycin, ⁇ -mercaptoethanol, such as DMEM / F12 medium It can be maintained using Primate ES medium (Reprocell).
- the differentiation induction of nephron progenitor cells from pluripotent stem cells such as ES cells or iPS cells in the present invention includes both a culture system containing feeder cells and a feeder-free culture system.
- a medium used for differentiation induction a commonly used medium can be used, and there is no particular limitation as long as the object of the present invention can be achieved.
- a medium used for culturing animal cells can be prepared as a basal medium.
- basal medium for example, BME medium, BGjB medium, CMRL 1066 medium, Glasgow MEM medium, improved MEM medium, IMDM medium, Medium 199 medium, Eagles MEM medium, ⁇ MEM medium, DMEM medium, ham medium, RPMI 1640 medium, Fischer Examples thereof include 's medium, Dulbecco medium, improved Dulbecco medium, and mixed media thereof.
- a mixed medium of Iskov modified Dulbecco medium and Ham F12 can be used for induction of differentiation of ES cells
- DMEM / F12 medium can be used for induction of differentiation of iPS cells. Not limited.
- the medium used in the culture method of the present invention may be a serum-containing medium or a serum-free medium, but a serum-free medium is preferable from the viewpoint of ensuring the safety of cell transplantation by eliminating different components.
- the serum-free medium means a medium that does not contain unconditioned or unpurified serum, and is mixed with purified blood-derived components, animal tissue-derived components (for example, growth factors), or serum substitutes.
- the existing medium shall correspond to a serum-free medium.
- examples of such serum-free medium include serum-free medium supplemented with an appropriate amount (for example, 1-20%) of commercially available KSR, serum-free medium supplemented with insulin and transferrin, medium supplemented with cell-derived factors, and the like. However, it is not limited to these.
- differentiation induction of nephron progenitor cells from pluripotent stem cells can be performed using a medium in which each component or factor is added to the above medium in each step according to the present invention.
- the components and factors added to the medium are not limited to these, and examples thereof include B27, N2, insulin-transferrin-serenium, ⁇ -mercaptoethanol, ascorbic acid, and non-essential amino acid.
- the term “differentiation” or “differentiation induction” of the pluripotent stem cell referred to in the present invention is used in the meaning including that the pluripotent stem cell is induced to differentiate to the intermediate mesoderm and further to the metanephric nephron progenitor cell, Are also used in the sense that they are induced to differentiate into a three-dimensional kidney containing tubules and glomeruli.
- FIG. 1 shows an outline of the whole process of inducing nephron progenitor cells from ES cells using the present invention
- FIG. 2 shows an outline of the whole process of inducing nephron progenitor cells from iPS cells.
- nephron progenitor cells When nephron progenitor cells are induced to differentiate from mammal-derived pluripotent stem cells using the present invention, it is necessary to include the following three steps (a) to (c) in that order: ) Culturing embryoid bodies derived from pluripotent stem cells in a medium containing Bmp and a high concentration (concentration A) Wnt agonist; step (b) said embryoid bodies with Bmp and medium concentration (concentration) B) culturing in a medium containing a Wnt agonist of B; and step (c) culturing the embryoid body in a medium containing Fgf and a low concentration (concentration C) of a Wnt agonist.
- Bmp used in the steps (a) and (b) is selected from the group consisting of Bmp family such as Bmp1, Bmp2, Bmp4, Bmp6, Bmp7, Bmp8a, Bmp8b and Bmp10, preferably selected from Bmp2, Bmp4 or Bmp7 More preferably, it is Bmp4.
- the Fgf used in the step (c) is selected from the Fgf family such as Fgf2, 9, 20, etc., preferably selected from Fgf2, Fgf9 or Fgf20, more preferably Fgf9.
- concentration of the Wnt agonist used in steps (a) to (c) is concentration A> concentration B> concentration C, and concentration A is at least 5 times the concentration C.
- the method of the present invention includes these steps in the order of (a), (b), and (c), and may include another step between the steps as necessary. a), (b) and (c) are included in succession. By continuing these steps, nephron progenitor cells can be efficiently differentiated from pluripotent stem cells.
- the medium further contains activin, more preferably activin and retinoic acid.
- the medium is a medium containing neither Bmp, activin nor retinoic acid.
- the embryoid body used in the step (a) can be prepared by culturing pluripotent stem cells (for example, ES cells or iPS cells) in any medium, preferably a serum-free medium.
- pluripotent stem cells for example, ES cells or iPS cells
- embryoid bodies are preferably prepared using mouse ES cells, and more preferably treated with activin before the treatment of step (a).
- the concentration of activin in the medium can be, for example, 0.1 to 3 ng / mL, preferably 0.5 to 1 ng / mL.
- the treatment with activin can be performed, for example, for 1 to 2 days, preferably 1 day.
- Bmp4 when Bmp4 is used in a combination of about 0.1 ng / ml to 0.3 ng / ml, differentiation induction efficiency may increase.
- human pluripotent stem cells when used, the present invention is not limited thereto.
- human iPS cells are treated with Bmp4 and a Rock inhibitor (Y27632) (added with Fgf as necessary).
- the embryoid body is prepared, and more preferably, the embryoid body treated with activin and Fgf is then 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.
- the concentration of the Rock inhibitor (Y27632) in the medium is, for example, 1 to 100 ng / mL, preferably 5 to 20 ng / mL can be raised, and the concentration of Fgf in the medium can be raised, for example, 0 to 20 ng / mL.
- the concentration of activin in the medium is, for example, 0 1 to 5 ng / mL, preferably 0.5 to 1 ng / mL.
- the treatment with Bmp4 and Rock inhibitor (Y27632), Fgf can be carried out, for example, for 1 to 2 days, preferably 1 day, and the treatment with activin and Fgf can be carried out, for example, for 1 to 4 days, preferably 2 days. .
- the concentrations A, B and C of the Wnt agonist contained in the medium used in each step have a specific relationship. This is very important.
- the concentration of Wnt agonist in the medium used in each step is concentration A> concentration B> concentration C and concentration A is at least 5 times the concentration C, but preferably concentration A is at least twice concentration B
- 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 in the method of the present invention, and is appropriately selected depending on the Wnt agonist used.
- CHIR99021 when CHIR99021 is used, it is 0. 1 to 3.0 ⁇ M, preferably 0.5 to 2.0 ⁇ M.
- the concentration A is 6 to 20 ⁇ M, preferably 7 to 15 ⁇ M, more preferably as a combination of Wnt agonist concentrations A and C in steps (a) and (c).
- a combination selected from 10 ⁇ M and a concentration C selected from 0.5 to 2 ⁇ M, preferably 0.7 to 1.5 ⁇ M, more preferably 1 ⁇ M can be mentioned.
- concentration A is preferably 6 to 20 ⁇ M, preferably Is selected from 7 to 15 ⁇ M, more preferably 10 ⁇ M
- concentration B is selected from 2 to 6 ⁇ M, preferably 2 to 4.5 ⁇ M, more preferably 3 ⁇ M
- concentration C is 0.5 to 2 ⁇ M, preferably 0
- a combination selected from 7 to 1.5 ⁇ M, more preferably 1 ⁇ M can be mentioned.
- the Wnt agonist that can be used in the present invention is not particularly limited as long as it has Wnt agonist activity.
- a Wnt agonist is defined as an agent that activates TCF / LEF-mediated transcription in a cell.
- Wnt agonists are selected from true Wnt agonists, inhibitors of intracellular ⁇ -catenin degradation and activators of TCF / LEF that bind to and activate Frizzled receptor family members, including any and all of the Wnt family proteins.
- a Wnt agonist is at least 10%, preferably at least 30%, more preferably at least 50%, even more preferably at least 70% compared to the level of Wnt activity in the absence of this molecule, Even more preferably at least 90%, most preferably 100% refers to those that stimulate Wnt activity in the cell.
- Wnt activity can be determined by measuring the transcriptional activity of Wnt, for example, by pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (Korinek et al., Science 275: 1784-1787, 1997).
- the Wnt agonists that can be used in the present invention include Wnt-1 / Int-1; Wnt-2 / Irp (Int-1 related protein); Wnt-2b / 13, Wnt-3 / Int-4; Wnt-3a; Wnt-4; Wnt-5a; Wnt-5b; Wnt-6; Wnt-7a; Wnt-7b, Wnt-8a / 8d; Wnt-8b; Wnt-9a / 14; Wnt-9b / 14b / 15; 10a; Wnt-10b / 12; including secreted glycoproteins including Wnt-11 and Wnt-16.
- Wnt agonists function as Wnt proteins in that they bind to the R-spondin family of secreted proteins and the Frizzled-4 receptor with high affinity and induce activation of the Wnt signaling pathway.
- Wnt agonists are small molecule agonists of the Wnt signaling pathway, aminopyrimidine derivatives.
- Wnt agonists included in the above definition also include Wnt signaling pathway inhibitors, GSK-3 inhibitors, Dkk1 antagonists and the like.
- a GSK-3 inhibitor includes a GSK- ⁇ or ⁇ inhibitor, and is defined as a substance that inhibits the kinase activity of GSK-3 ⁇ or ⁇ protein, for example, the ability to phosphorylate ⁇ -catenin. Many substances are known. ing.
- CHIR99021 (6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] Amino] nicotinonitrile), lithium, valproic acid, benzazepineone family of kenpaulone; 9-bromo-7,12-dihydroindolo [3,2-d] [1] benzacepine-6 (5H) -One), Alsterpaulone (9-nitro-7,12-dihydroindolo [3,2-d] [1] benzacepin-6 (5H) -one), 5-chloro-indirubin which is an indirubin derivative , Indirubin-3'-monooxime and BIO (aka GSK-3 ⁇ inhibitor IX; 6-bromoindirubin-3′-oxime), a maleimide derivative SB216673 (3- (2,4-dichlor
- the Wnt agonist that can be used in the present invention further contains a Wnt signal transduction pathway inhibitor, and a substance known or marketed as a Wnt signal transduction pathway inhibitor can be used.
- the Wnt agonist that can be used in the present invention includes any natural product or synthetic product as long as it is included in the above definition, and may be any protein, polymer, or small molecule.
- Examples of Wnt agonists that can be used in the present invention include, but are not limited to, for example, preferably GSK-3 inhibitors, more preferably CHIR99021, BIO, or SB415286, particularly preferably. CHIR99021.
- the use concentration of each Wnt agonist can be appropriately selected according to the purpose of use, and for example, a concentration that can exhibit the same effect as that obtained when CHIR99021 is used can be selected.
- Bmp used in step (a) can be any of the above-described Bmp, and the concentration of each Bmp compound used can be appropriately selected according to the purpose of use.
- Bmp4 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 an effect of inducing differentiation is obtained.
- the concentration at which the same effect as that obtained when Bmp4 is used can be appropriately selected.
- any of the above-described Bmp can be used as the Bmp used in the step (b), and the use concentration of each Bmp compound can be appropriately selected according to the purpose of use.
- Bmp4 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 an effect of inducing differentiation is obtained. -30 ng / mL, preferably 1-10 ng / mL.
- the concentration that can achieve the same effect as that obtained when Bmp4 is used can be appropriately selected.
- the activin used in step (b) is any However, human activin A is preferred.
- the concentration in the medium is not particularly limited as long as the differentiation-inducing effect is obtained, and can be, for example, 2.5 to 40 ng / mL, preferably 7.5 to 15 ng / mL.
- the concentration of retinoic acid used in the step (b) in the medium is not particularly limited as long as an effect of inducing differentiation is obtained, and is, for example, 0.001 to 1 ⁇ M, preferably 0. 01-0.3 ⁇ M can be raised.
- any of the above-described Fgf can be used, and the use concentration of each Fgf compound can be appropriately selected according to the purpose of use.
- Fgf9 when Fgf9 is used, Fgf9 of any origin can be used, but human Fgf9 is preferred.
- the concentration in the medium is not particularly limited as long as the differentiation-inducing effect is obtained, and can be, for example, 1 to 25 ng / mL, preferably 2.5 to 10 ng / mL.
- concentration which can exhibit the effect similar to the effect acquired when Fgf9 is used can be selected suitably.
- the number of days to be treated in the steps (a), (b) and (c) is not particularly limited as long as nephron progenitor cells can be induced, but there are preferable days for mice and humans as follows.
- the culture is performed, for example, in the step (a) for 1 to 4 days, preferably 2 to 3 days, particularly preferably 2.5 days.
- Step (b) is preferably carried out for 0.5 days to 2 days, particularly preferably 1 day
- step (c) is carried out, for example, for 0.5 to 3 days, preferably 1 to 2.5 days, in particular Preferably it can be performed for 2 days.
- Step (b) is preferably carried out for 1 to 3 days, particularly preferably 2 days
- step (c) is carried out, for example, for 1 to 5 days, preferably 2 to 4 days, particularly preferably 3 days.
- the nephron progenitor cell induced by the method of the present invention is a cell population that expresses all transcription factors Osr1, Wt1, Pax2, Six2, Hoxa10, and Hoxa11 that define the metanephric mesenchyme, and their genes Are co-expressed at a single cell level with high probability.
- the nephron progenitor cells of the present invention are metanephric nephron progenitor cells that can reconstitute not only tubules but also three-dimensional kidneys containing glomeruli.
- proximal tubular cell a cell population of cells that express Cadherin 6, Megalin, and LTL.
- Distal tubular cells are characterized by a cell population of cells that express E-cadherin, Brn1, and NCC.
- a characteristic of glomerular cells is the cell population of cells that express Wt1, Nephrin, and Podocin.
- Proximal tubular cells, distal tubular cells, or glomerular epithelial cells can be obtained by co-culturing nephron progenitor cells with, but not limited to, fetal spinal cord or Wnt4 expressing cells, for example.
- the sorting of each constituent cell after co-culture is not limited to this, but for example, after cell dissociation treatment using trypsin or the like, each cell-specific membrane protein (for example, podocalyxin in the case of glomerular epithelial cells, Cadherin 6 for the proximal tubule and Ecadherin for the distal tubule can be antibody-stained and fractionated using a FACS (flow cytometer).
- Another aspect of the present invention is a method for producing a three-dimensional kidney in which tubules and glomeruli are reconstructed using nephron progenitor cells obtained by the method of the present invention.
- the method for producing a three-dimensional kidney using nephron progenitor cells obtained by the method of the present invention can be performed, for example, by co-culturing nephron progenitor cells with embryonic spinal cord or Wnt4-expressing cells at the gas-liquid interface. it can.
- the conditions of co-culture can be performed with reference to the method described in Kispert et al., Development 125, 4225-4234, 1998 (non-patent document 8), but are not limited thereto, and other known findings Basically, those modified or improved appropriately by those skilled in the art are also included.
- the kidney thus created forms a three-dimensional kidney structure including tubules and glomeruli.
- Another embodiment of the present invention is a three-dimensional kidney in which tubules and glomeruli prepared using nephron progenitor cells obtained by the method of the present invention are reconstituted.
- the method for creating a three-dimensional kidney is as described above.
- Another embodiment of the present invention is a medium and a medium kit for inducing nephron progenitor cells from pluripotent stem cells.
- One characteristic of the medium of the present invention is a combination of mediums containing Wnt agonists whose concentrations are changed stepwise, and the present invention is also a medium kit for making such medium combinations.
- a medium kit comprising an induction medium for inducing nephron progenitor cells or kidneys from pluripotent stem cells and a Wnt agonist can be used, and at the time of use, a Wnt agonist is added to the medium so as to have a stepwise concentration. Is done.
- the culture medium kit of the present invention may further contain any one or more of Bmp4, activin, and retinoic acid.
- the present invention made it possible to induce metanephric nephron progenitor cells from pluripotent stem cells.
- the metanephric nephron progenitor cells generated by the method of the present invention can contribute to the construction of more mature nephron components, and can combine them with ureteric bud-derived structures to confer renal physiological function. It becomes possible.
- the target vector was electroporated into E14.1 ES cells and then three 480G418 resistant clones were determined as targets by digestion with BamHI followed by Southern blot analysis using a 5 ′ probe. Correctly targeted ES clones were used to create germline chimeras that mated with C57BL / 6J female mice. When Neo was removed by mating Osr1-GFP mutant mice with mice ubiquitously expressing Cre, the phenotype and EGFP expression pattern were identical to those of the original mutant mice.
- CDB0604K http://www.cdb.riken.jp/arg/mutant%20mice%20list.html ), Wt1 tm1 (EGFP / cre) Wtp mouse, Six2 tm3 (EGFP / cre / ERT2) Amc mice and Gt (ROSA) 26 Sort9 (CAG-tdTomato) Hze mice were purchased from Jackson Laboratory (USA). All animal experiments were performed according to institutional guidelines and ethics committees.
- the cells were then transferred to 5% knockout serum replacement (Invitrogen), 10 ⁇ g / mL insulin, 6.7 ⁇ g / L sodium selenite, 5.5 ⁇ g / mL transferrin, 1 ⁇ 10 ⁇ 7 mol / L dexamethasone, 10 mmol / L.
- the cells were cultured in DMEM / F12 containing nicotinamide, 2 mmol / L L-glutamine, 50 ⁇ M / L 2-mercaptoethanol, 5 mmol / L HEPES and penicillin / streptomycin.
- Nuclei were counterstained with DAPI (Roche).
- DAPI DAPI
- the sample was fixed with 4% paraformaldehyde and embedded in an optimal cutting temperature (OCT) compound (Tissue Tek) to obtain a frozen section having a thickness of 10 ⁇ m.
- OCT optimal cutting temperature
- the sections were incubated in blocking solution. The subsequent procedure was the same as that for staining paraffin sections.
- T-GFP + cells were selected by FACS.
- the sorted cells were aggregated in a 96-well low cell adhesion plate at 7,000 cells per aggregate, and cultured in a serum-free known composition medium.
- the tail region from the 23-segment area of 22-26 somite embryos was collected and Osr1-GFP + or Wt1-GFP + cells were sorted by FACS. Sorted cells were aggregated at 10,000 cells per aggregate in a 96-well low cell adhesion plate and cultured in serum-free known composition medium.
- Mouse ES cell and human iPS cell culture Mouse ES cells (Osr1-GFP) were mixed with 15% fetal bovine serum, 0.1 mM 2-mercaptoethanol (Nacalai Tesque) and 1,000 U / mL leukemia inhibitory factor ( ESGRO) was maintained on mouse embryonic fibroblasts in DMEM (Invitrogen) supplemented.
- EB3-DsRed cells were a gift from Dr. Hitoshi Niwa (RIB CDB). EB3-DsRed cells were maintained as reported by Usui et al. (Am J Pathol 180, 2417-2426, 2012).
- ES cells were treated with DMEM supplemented with 15% fetal calf serum, 0.1 mM 2-mercaptoethanol, 1,000 U / mL leukemia inhibitory factor, 3 ⁇ M CHIR99021 (Wako) and 1 ⁇ M PD0325901 (Wako). Invitrogen) for one passage on gelatin-coated dishes without feeder cells.
- Differentiation of ES cells was performed in a serum-free medium as follows. ES cells were dissociated with Accutase (ESGRO) and 0.5% N2 and 0.5x B27 (without retinoic acid) in 75% Iskov modified Dulbecco medium (Invitrogen) and 25% Ham F12 medium (Invitrogen).
- ESGRO Accutase
- N2 and 0.5x B27 without retinoic acid
- EBs embryoid bodies
- the medium was switched to BC10 medium containing 1 ng / mL human Bmp4 (R & D Systems) and 10 ⁇ M CHIR99021.
- the medium was changed to fresh medium (BC10).
- the medium was changed to ABC3R medium containing 10 ng / mL activin, 3 ng / mL Bmp4, 3 ⁇ M CHIR99021 and 0.1 ⁇ M retinoic acid.
- the medium was changed to C1F medium containing 1 ⁇ M CHIR99021 and 5 ng / mL human Fgf9 (R & D Systems).
- Human iPS cells (201B7) were maintained on mouse embryonic fibroblasts in Primate ES medium (Reprocell) supplemented with 5 ng / mL recombinant human basic Fgf (Wako). On the third day of culture, colonies of iPS cells were detached and collected in 1 mg / ml Type 4 collagenaseit (Invitrogen). Furthermore, in order to remove mouse embryo fibroblasts, the collected cell suspension was allowed to stand for 10 minutes, and only iPS cell colonies were collected. Differentiation of iPS cells was performed in a serum-free medium as follows.
- iPS cells were dissociated with Accutase (ESGRO), 2% (v / v) B27 (without retinoic acid), 2 mM L-glutamine, 1% (v / v) ITS, 1% (v / v) non-essential amino acids
- ESGRO Accutase
- 2% (v / v) B27 (without retinoic acid) 2 mM L-glutamine
- 1% (v / v) ITS 1% (v / v) non-essential amino acids
- the cells were cultured in a serum-free differentiation medium consisting of DMEM / F12 (Invitrogen) supplemented with 90 ⁇ M ⁇ -mercaptoethanol and 0.5 ⁇ penicillin / streptomycin (without retinoic acid).
- the collected cells were aggregated at 10,000 cells per aggregate in a 96-well low cell adhesion plate in the presence of 10 ⁇ M Y27632 (Wako) and 0.5 ng / mL human Bmp4 (R & D Systems) Embryoid bodies (EBs) were formed.
- the medium was changed to a mesoderm induction medium containing 1 ng / mL human activin and 20 ng / mL human basic Fgf (R & D Systems).
- the medium was switched to BC10 medium containing 1 ng / mL human Bmp4 (R & D Systems) and 10 ⁇ M CHIR99021.
- Non-patent Document 8 Development 125, 4225-4234, 1998) and Osafune et al.
- Non-patent Document 4 Development 133, 151-161, (2006), mouse embryonic metanephric mesenchymal cells or induced ES cell aggregates were air-liquid on a polycarbonate filter (0.8 ⁇ m, Whatman) using DMEM containing 10% fetal bovine serum. At the interface, cultured with embryonic spinal cord taken from E11.5 or E12.5 embryos or on 3T3Wnt4 cells.
- Antibody The following antibodies were used: rabbit anti-Pax2 (Covance; 1: 800); fluorescein anti-LTL (FL-1321; Vector Laboratories; 1: 100); chicken anti-GFP (Abcam; 1: 1000) ); Rabbit anti-GFP (Invitrogen; 1: 400); Rabbit anti-Itga8 (Sigma; 1: 200); Rabbit anti-Pdgfra (Cell Signaling Technology; 1: 500); Mouse anti-Pdgfra (Takakura et al., J Histochem Cytochem 45, 883) -893, 1997) (1: 500); rabbit anti-Wt1 (Santa Cruz Biotechnology; 1: 200); mouse anti-Wt1 (Dako; 1: 100); rabbit anti-Six2 (Proteintech; 1: 500); Sall1 (PPMX Perseus Proteomics; 1: 200); mouse anti-E-cadherin (BD Biosciences; 1: 800); rabbit anti-Cdh6 (
- Dressler Cho et al., Development 125, 803-812, 1998); 1: 400); mouse anti-Aqp1 (Abcam; 1: 100); rabbit anti-Podocin (provided by Dr. Kakinuma (Lydia et al., Am J Nephrol 35, 58-68., 2012); 1: 400); guinea pig anti-Nephrin (Progen; 1: 200); rabbit anti-CD31 (Abcam; 1:25); rat anti-CD34 (Abcam; 1: 100); rabbit anti-DsRed (Clontech; 1: 100).
- Quantitative RT-PCR RNA was isolated using RNeasy Plus Micro Kit (Qiagen) and then reverse transcribed using random primers and Superscript III (Invitrogen). Quantitative PCR was performed using Real-Time PCR System (Applied Biosystems) and Thunderbird SYBR qPCR Mix (Toyobo). All samples were normalized by ⁇ -actin expression using the relative standard curve method.
- Microarray analysis was performed using an Agilent SurePrint G3 mouse gene expression (8 ⁇ 60K) microarray. Data was standardized by GeneSpring GX software (Agilent). Microarray data was deposited with the National Center for Biotechnology Information Gene Expression Omnibus (GSE).
- GSE National Center for Biotechnology Information Gene Expression Omnibus
- Example Example 1 Osr1 + / Integrina8 + / Pdgfra-population metanephric mesenchyme representing colony-forming progenitor cells , glomerular epithelium as shown by cell fate analysis comprising labeling mesenchyme expressing transcription factor Six2 (Including podocytes) and the tubules that make up the main part of the nephron.
- the inventors have previously demonstrated the presence of nephron progenitor cells by establishing a novel colony formation assay.
- Non-patent document 9 Nishinakamura et al., Development 128, 3105-3115, 2001; Non-patent document 4: Osafune et al., Development 133, 151-161, 2006).
- the Sall1 high and Six2 positive metanephric mesenchyme represents the nephron precursor population in the embryonic kidney.
- Osr1 is another metanephric mesenchymal marker and one of the earliest markers of the intermediate mesoderm.
- Non-Patent Document 10 James et al., Development 133, 2995-3004, 2006; Non-Patent Document 1: Mugford et al., Dev Biol 324, 88 -98, 2008. Therefore, an Osr1-GFP knock-in mouse was created (FIG. 3), and the green fluorescent protein (GFP) was E8.5-E9.5 intermediate mesoderm and E11.5-E15.5 metanephric mesenchyme. (Figs. 4A to 4C).
- OSR1-GFP positive population contains colony-forming nephron progenitor cells.
- the tail part of E8.5 embryo (from heart level to tail) and E9.5 embryo (from forelimb level to tail) were collected.
- E11.5 and E15.5 embryonic metanephros were dissected manually.
- OSR1 + cells were sorted by FACS and seeded on Wnt4 feeder cells. On day 8, the number of colonies was counted. The results are shown in Table 1 below.
- the OSR1-GFP positive population selected from E11.5 and E15.5 embryonic kidneys contained colony-forming nephron progenitor cells.
- E9.5 or E11.5 sections were collected.
- E10.5 the middle kidney region (front forelimb end to front hindlimb end) or hindrenal region (front hindlimb end to back hindlimb end) was dissected manually.
- the collected tissues were separated and immunostained with anti-Itga8 antibody and anti-Pdgfra antibody.
- OSR1 + / Itga8 + / Pdgfra ⁇ cells were sorted by FACS and seeded on Wnt4 feeder cells. On day 8, the number of colonies was counted. The results are shown in Table 2 below. As described below, colony-forming nephron progenitor cells were enriched in the Osr1 + / Itga8 + / Pdgfra ⁇ fraction.
- Example 2 Anterior intermediate mesoderm at E9.5 contains colony-forming progenitor cells that contribute to the mid-kidney
- the expression of nephron progenitor cell markers and the colony-forming ability of Osr1-GFP positive cells at the early stage were examined. did.
- FIG. 5 and Table 1 in E8.5, no overlap of Itga8 and GFP was detected and colony formation by GFP + population was not detected, but in E9.5, colony formation by GFP + population was detected ( 0.037 ⁇ 0.013%). Also, colony-forming cells were enriched by finding the GFP + region that was Itga8 + / Pdafra ⁇ (FIG.
- colony-forming precursor cells were detected only in the front part of GFP + cells in the E9.5 embryo. While Wt1 (another nephron progenitor cell marker) is expressed in both the front part and the tail part, as shown in FIG. 11, the markers for nephron progenitor cells, Pax2 and Six, are mainly in the middle It was expressed in the front of the germ layer. These data suggest a molecular difference between the anterior and tail intermediate mesoderm at E9.5. Furthermore, the mouse having the tdTomato reporter allele and the Six2-GFPCreER mouse were crossed, and tamoxifen was injected into Cre that was temporarily activated at E9.5. When analyzed at E11.5, as shown in FIG.
- Example 3 Induction of metanephric nephron progenitor cells from the tail middle mesoderm of E9.5 E9.5 mid-renal progenitor cells estimated to be Osr1 + / Itga8 + / Pdgfra-, and E10.5-11 .5 metanephric nephron progenitor cells were used for microarray and quantitative PCR analysis. The results are shown in FIG. 13 and FIG. Both types of progenitor cells express common transcription factors such as, for example, Osr1, Wt1, Pax2 and Six2, and Gdnf (cytokine essential for kidney development), while the metanephric progenitor cells are Hoxa10, Hoxa11.
- Hox11 family genes that begin to be expressed at the posterior end of the embryo around E9.0 have been reported to be essential for metanephric development by determining the metanephric region along the anteroposterior axis of the intermediate mesoderm.
- cell fate mapping studies indicate that E9.5 Osr1 + intermediate mesoderm contributes to the metanephric mesenchyme. Therefore, we hypothesized that non-colony forming Osr1 + / tail Hox + intermediate mesoderm located posteriorly at E9.5 could be a precursor population of metanephric nephron progenitor cells (FIGS. 15, 11 and 11). FIG. 13).
- Osr1-GFP + cells were selected from the tail portion of E9.5 embryos and plated on low cell adhesion plates in the presence of Rho kinase inhibitor Y27632, which supports cell survival. It was. The results are shown in FIG. The cells reaggregated spontaneously and formed spheres within 24 hours. At 48 hours of culture, the aggregates had a strong GFP signal and showed Pax2, Six2 and Gdnf expression more than 10 times higher than at the start of culture. In addition, as shown in FACS analysis (FIG. 17), the appearance of Osr1 + / Itga8 + / Pdgfra ⁇ population was shown (10.0 ⁇ 0.01% of all cells). When these aggregates were separated and stimulated with Wnt4, colony formation was observed as shown in FIG. This suggests in vitro induction of metanephric nephron progenitor cells from the tail middle mesoderm.
- Non-Patent Document 11 Barak et al., Dev Cell). 22, 1191-1207., 2012, and Non-Patent Document 12: Poladia et al., Dev Biol 291, 325-339, 2006).
- Example 4 The metanephric mesenchymal precursor is maintained in a T-positive tail population until the E8.5 post gastrulation stage, followed by an early stage mesoderm in the E9.5 tail middle mesoderm method The in vitro method for differentiating into renal mesenchyme was examined.
- One report shows that both metanephric mesenchyme and ureteric bud derived from intermediate mesoderm appear around embryonic (E) 8.5 and express transcription factor Osr1 (Non-patent Document 1). : Mugford et al., Dev Biol 324, 88-98, 2008).
- Non-patent document 13 Atsuta et al., Dev Growth Differ 55, 579-590, 2013,
- Non-patent document 14 Attia et al., Development 139, 4143-4151, 2012
- Non-patent document 15 Obara-Ishihara et al. Development 126, 1103-1108, 1999
- Non-Patent Document 16 Saxen, Cell 131, 861-872, 1987).
- the E8.5 Pax2 / 8-positive pre-mesoderm (referred to as the prorenal primordium) is presumed to be an equivalent population and is included in the Osr1-positive region. Therefore, we first examined the effects of many combinations of growth factors using sorted E8.5 Osr1-GFP + cells. However, colony forming progenitor cells could not be induced.
- T a representative marker of primitive streak and undifferentiated mesoderm of the tail, causes shortening of the tail including the metanephric region
- Non-patent Document 17 a mouse having a tdTomato reporter allele and a TnEGFP-CreERT2 / + mouse were crossed, and tamoxifen was injected in the gastrulation phase (E6.5 and E7.5) when early germ layer formation occurred.
- E6.5 and E7.5 gastrulation phase
- tamoxifen injection at E8.5 revealed that labeled cells were detected only in the lower trunk of the E11.5 embryo containing the hindlimb mesenchyme located at the hindlimb level.
- the heart and forelimb which are mesoderm tissues located in front, were no longer labeled.
- the metanephric mesenchyme, not the ureteric bud was labeled when the metanephric section was made.
- Example 5 T-tailed nephron progenitor cells were induced from T-positive tail mesoderm in E8.5 using T + tail mesoderm of E8.5 selected as a raw material based on the hindline mesenchymal induced line tracing experiment . The results are shown in FIG. Sorted cells were reaggregated to form aggregates, treated with various growth factors, and then treated with C1F as described above (FIG. 22C, step 3). Since the posterior intermediate mesoderm is identified by the expression of Osr1, Wt1 and tail Hox genes, we first focused on growth factors that may affect the expression of these genes.
- Bmp and Wnt signaling have been reported in the expression of the tail Hox gene in mouse embryonic stem (ES) cell differentiation, and retinoic acid signaling is important for Wt1 cognate expression in zebrafish development It has been reported. Since co-introduction of retinoic acid, Bmp and Wnt agonists did not increase the tail Hox gene to the level observed in the embryonic post-renal mesenchyme (FIGS. 22D and E), an “retrophase” was added. That is, since a canonical Wnt signal has been reported to be important for trunk elongation, a Wnt agonist was added at a high concentration (10 ⁇ M CHIR) in combination with Bmp4.
- Example 6 Induction of metanephric nephron progenitor cells from mouse ES cells Induction of metanephric nephron progenitor cells from ES cells was performed. An overview of the overall process of induction of the metanephric mesenchyme from ES cells is shown in FIG. 1, and the expression of the signature gene at each stage is shown in FIG. In order to monitor the induction of intermediate mesoderm and metanephric nephron progenitor cells, embryos were cultured in serum-free medium without factors for 2 days using the Osr1-GFP ES cell line prepared from the Osr1-GFP mice described above. Forms (EBs) were created.
- EBs Forms
- EBs metanephric nephron progenitor cells were prepared. For the next 24 hours, a low concentration of activin was added to induce transient expression of the embryonic ectoderm marker Fgf5. EBs were then treated with Bmp4 and high concentration of CHIR (step 2). At 4.5 days, the expression of T, a neoplastic precursor, and the expression of Cdx2 and Tbx6 were upregulated. Subsequently, the protocol for the posterior mesoderm described above could be fully reproduced (FIG. 22C and steps 3-5 of FIG. 1).
- Induced EBs harvested at day 8.5 expressed multiple signature genes for metanephric nephron progenitor cells at levels comparable to embryonic retrorenal mesenchyme (FIG. 25).
- the results of immunostaining showed that many cells co-expressed typical renal transcription factors including Osr1, Wt1, Pax2, Sall1 and Six2 (FIG. 26).
- the results of FACS analysis showed that nearly 90% of the cells were Osr1-GFP positive, and Itga8 + / Pdgfra-progenitor cells comprised about 65% of the Osr1 + cells (FIG. 27).
- Example 7 Formation of three-dimensional kidney structure using ES cells
- the metanephric mesenchyme from E11.5 embryos undergoes mesenchymal epithelial transformation when co-cultured with embryonic spinal cord or Wnt4 expressing cells at the gas-liquid interface, It is well established to form glomeruli and tubules (Non-patent Document 8: Kispert et al., Development 125, 4225-4234, 1998).
- the glomeruli and tubules created using E11.5 embryos are shown in FIG. 28A. Therefore, when the EBs induced in Example 6 were cultured by the same method, firm tubule formation was confirmed. The results are shown in FIGS. 28C and D.
- FIG. 28E histological examination of EBs harvested with day 6 showed a number of tube formations under any condition (FIGS. 28C and D), which was confirmed by E-cadherin staining (FIG. 28E).
- Most of the tubes are positive for Pax2 and Sall1, indicating renal tubules (FIGS. 28E-H, LP).
- Some tubules expressed proximal tubule markers, such as LTL or cadherin-6, aquaporin 1, Jagged 1, Megalin, etc. (FIGS. 28F, G, L-N).
- Example 8 Induction of metanephric nephron progenitor cells from human iPS cells The protocol used for mouse ES cells described above was applied to human iPS cells to differentiate iPS cells toward metanephric nephron progenitor cells in vitro. . An overview of the overall process of induction of the metanephric mesenchyme from iPS cells is shown in FIG. Previous reports have shown that Bmp, Fgf and activin signals are important for early induction of cells of the mesodermal lineage in human pluripotent stem cells (Non-patent Document 19: Bernard et al.
- pluripotent stem cells such as ES cells and iPS cells can be induced to differentiate into metanephric nephron progenitor cells.
- the present invention can also be used as a part of the process in the formation of a three-dimensional kidney structure from pluripotent stem cells. Therefore, the present invention is useful in research and regenerative medicine using differentiation induction from pluripotent stem cells to the kidney.
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
(1) 哺乳動物由来の多能性幹細胞から(後腎)ネフロン前駆細胞である後腎間葉を分化誘導する方法であって、該方法が、以下の3つの工程を、
(a)多能性幹細胞から誘導された胚様体を、Bmpおよび高濃度(濃度A)のWntアゴニストを含有する培地で培養する工程、
(b)前記胚様体を、Bmpおよび中濃度(濃度B)のWntアゴニストを含有する培地で培養する工程、および
(c)前記胚様体を、Fgfおよび低濃度(濃度C)のWntアゴニストを含有する培地で培養する工程、
をその順で含むことを特徴とする分化誘導方法
(ここで、Wntアゴニストの濃度は、濃度A>濃度B>濃度Cであって、濃度Aは濃度Cの少なくとも5倍である。)。
(2)前記工程(a)、(b)および(c)におけるWntアゴニストの濃度は、濃度Aは濃度Bの少なくとも2倍(好ましくは少なくとも3倍)であり、かつ濃度Bは濃度Cの少なくとも2倍(好ましくは少なくとも3倍)である、前記(1)に記載の分化誘導方法。
(3) 前記工程(b)において、培地がさらにアクチビンを含む、前記(1)または(2)に記載の分化誘導方法。
(4) 前記工程(b)において、培地がさらにレチノイン酸を含む、前記(3)に記載の分化誘導方法。
(6) 前記Wntアゴニストが、CHIR99021、BIO、およびSB415286からなる群より選ばれる(ただし、各工程のWntアゴニストは同じであっても異なってもよい)、前記(5)のいずれか一つに記載の分化誘導方法。
(7) 前記Bmpが、Bmpファミリーからなる群、好ましくはBmp2、Bmp4およびBmp7からなる群より選ばれる、かつ、前記Fgfが、Fgfファミリーからなる群、好ましくはFgf2、Fgf9およびFgf20からなる群より選ばれる、前記(1)~(6)のいずれかに記載の分化誘導方法。
(8) 前記BmpがBmp4であり、かつ前記FgfがFgf9である、前記(1)~(7)のいずれかひとつに記載の分化誘導方法。
(9) 前記工程(a)、(b)および(c)におけるWntアゴニストが、CHIR99021であり、かつ、前記濃度Aが7.5μM~15μM、前記濃度Cが0.5μM~2.0μMである、前記(1)~(8)のいずれか一つに記載の分化誘導方法
(10) 前記工程(a)および(b)におけるBmpがBmp4であり、工程(a)における濃度が0.1ng/ml~3ng/mlであり、工程(b)における濃度が1ng/ml~10ng/mlである、前記(9)に記載の分化誘導方法。
(11) 前記工程(b)において、アクチビンを2.5~40ng/mLの濃度で含む前記(10)に記載の分化誘導方法。
(12) 前記工程(a)、(b)および(c)が、連続した工程である前記(1)~(11)のいずれか一つに記載の分化誘導方法。
(13) 前記工程(c)において、培地が、Bmp、レチノイン酸またはアクチビンのいずれも含まない培地である、前記(1)~(12)のいずれか一つに記載の分化誘導方法。
(15) 前記多能性幹細胞がヒトiPS細胞である、前記(14)に記載の分化誘導方法。
(16) 前記多能性幹細胞がマウスES細胞またはiPS細胞であり、かつ工程(a)が、少なくとも1日以上4日以内培養する工程(ここで、好ましくは、少なくとも1回新鮮な培地に交換を行う)である前記(14)に記載の分化誘導方法。
(17) 前記多能性幹細胞がヒトES細胞またはiPS細胞であり、かつ工程(a)が、少なくとも3日以上11日以内培養する工程(ここで、好ましくは、少なくとも2回新鮮な培地に交換を行う)である前記(14)に記載の分化誘導方法。
(18) 転写因子である、Osr1、Wt1、Pax2、Six2、Hoxa10、Hoxa11の全てを発現する細胞集団であることを特徴とする哺乳動物由来の多能性幹細胞から分化誘導されたネフロン前駆細胞。
(19) 前記多能性幹細胞が、マウスES細胞またはiPS細胞、またはヒトES細胞またはiPS細胞である、前記(18)に記載のネフロン前駆細胞。
(20) 前記(1)~(17)のいずれか一つに記載の分化誘導方法により誘導されたネフロン前駆細胞。
(21) 前記(18)~(20)のいずれか一つに記載のネフロン前駆細胞を用いて、糸球体および尿細管を有する三次元腎臓構造を作成する方法。
(22) 前記方法が、ネフロン前駆細胞を気液界面にて胚脊髄またはWnt4発現細胞と共培養することを含む、前記(21)に記載の三次元腎臓を作成する方法。
(23) 前記(21)または(22)に記載の方法により形成された、糸球体および尿管を有する三次元腎臓構造。
(24) 前記(18)~(20)のいずれか一つに記載のネフロン前駆細胞から分化誘導されたCadherin6、Megalin、およびLTLを発現する細胞集団であることを特徴とする近位尿細管細胞。
(25) 前記分化誘導が、ネフロン前駆細胞を胎児脊髄またはWnt4発現細胞と共培養することにより行われる、前記(24)に記載の近位尿細管細胞。
(26) 前記(18)~(20)のいずれか一つに記載のネフロン前駆細胞から分化誘導された、E-cadherin、Brn1、およびNCCを発現する細胞集団であることを特徴とする遠位尿細管細胞。
(27) 前記分化誘導が、ネフロン前駆細胞を胎児脊髄またはWnt4発現細胞と共培養することにより行われる、前記(26)に記載の遠位尿細管細胞。
(28) 前記(18)~(20)のいずれか一つに記載のネフロン前駆細胞から分化誘導された、Wt1、Nephrin、およびPodocinを発現する細胞集団であることを特徴とする糸球体上皮細胞。
(29) 前記分化誘導が、ネフロン前駆細胞を胎児脊髄またはWnt4発現細胞と共培養することにより行われる、前記(28)に記載の糸球体上皮細胞。
(30) 多能性幹細胞からネフロン前駆細胞を誘導するための分化誘導培地キットであって、該キットは、(i)分化誘導培地、および(ii)Wntアゴニストを含み、使用時において、前記Wntアゴニストを段階的濃度で含む少なくとも3つの分化誘導培地が調整される、ここで、前記段階的濃度は、少なくとも濃度A、BおよびCからなり、濃度A>濃度B>濃度Cであって、濃度Aは濃度Cの少なくとも5倍である、
ことを特徴とする培地キット、培地キット。
(31) 前記Wntアゴニストの段階的濃度は、濃度Aは濃度Bの少なくとも3倍であり、かつ濃度Bは濃度Cの少なくとも3倍であるように分化誘導培地が調整される、前記(30)に記載の培地キット。
ES細胞は、一般的には、胚盤胞期の受精卵をフィーダー細胞と一緒に培養し、増殖した内部細胞塊由来の細胞をばらばらにして、さらに、植え継ぐ操作を繰り返し、最終的に細胞株として樹立することができる。
iPS細胞の培養も定法により行うことができる。例えば、フィーダー細胞としてマウス繊維芽細胞を用いて、bFGF、KSR(ノックアウト血清代替物)、非必須アミノ酸、L-グルタミン、ペニシリン、ストレプトマイシン、β-メルカプトエタノールを加えた培地、例えばDMEM/F12培地やPrimate ES培地(リプロセル)を用いて維持することができる。
本発明を用いて哺乳動物由来の多能性幹細胞からネフロン前駆細胞を分化誘導する場合、以下の3つの工程(a)~(c)を、その順で含むことが必要である:工程(a)多能性幹細胞から誘導された胚様体を、Bmpおよび高濃度(濃度A)のWntアゴニストを含有する培地で培養する工程;工程(b)前記胚様体を、Bmpおよび中濃度(濃度B)のWntアゴニストを含有する培地で培養する工程;および工程(c)前記胚様体を、Fgfおよび低濃度(濃度C)のWntアゴニストを含有する培地で培養する工程。
工程(a)および(b)で用いられるBmpは、Bmp1、Bmp2、Bmp4、Bmp6、Bmp7、Bmp8a、Bmp8bおよびBmp10等のBmpファミリーからなる群より選ばれ、好ましくは、Bmp2、Bmp4またはBmp7から選ばれ、さらに好ましくはBmp4である。
工程(c)で用いられるFgfは、Fgf2,9,20等のFgfファミリーから選ばれ、好ましくはFgf2、Fgf9またはFgf20から選ばれ、より好ましくはFgf9である。
工程(a)~(c)で用いられるWntアゴニストの濃度は、濃度A>濃度B>濃度Cであって、濃度Aは濃度Cの少なくとも5倍である。
本発明の方法は、これらの工程を、(a)、(b)、(c)の順で含み、必要に応じて各工程の間に別の工程を含むこともできるが、好ましくは、(a)、(b)および(c)を連続で含む。これらの工程が連続することにより、多能性幹細胞からネフロン前駆細胞を効率よく分化誘導できる。
本発明の分化誘導方法においては、特に好ましくは、上記工程(c)において、培地がBmp、アクチビンまたはレチノイン酸のいずれも含まない培地である。
上記工程(a)により、後方中胚葉が誘導され、工程(b)により後方中間中胚葉が誘導され、そして工程(c)により後腎ネフロン前駆細胞(後腎間葉)が誘導される。
一方、ヒト多能性幹細胞を用いる場合は、これに限定されないが、好ましくはヒトiPS細胞を、Bmp4およびRock阻害剤(Y27632)で(必要に応じてさらにFgfを加えて)処理した細胞を用いて胚様体が調整され、さらに好ましくは、次いで、アクチビンおよびFgfで処理された胚様体が用いられる。培地中のBmp4の濃度は、例えば、0.3~5ng/mL、好ましくは0.5~2ng/mLをあげることができ、培地中のRock阻害剤(Y27632)の濃度は、例えば、1~100ng/mL、好ましくは5~20ng/mLをあげることができ、培地中のFgfの濃度は、例えば、0~20ng/mL、をあげることができ、培地中のアクチビンの濃度は、例えば、0.1~5ng/mL、好ましくは0.5~1ng/mLをあげることができる。Bmp4およびRock阻害剤(Y27632)、Fgfでの処理は、例えば、1~2日、好ましくは1日行い、アクチビンおよびFgfの処理は、例えば、1~4日、好ましくは2日行うことができる。
例えばCHIR99021を用いた場合、これに限定されないが、工程(a)および(c)におけるWntアゴニストの濃度AおよびCの組み合わせとして、濃度Aは、6~20μM、好ましくは7~15μM、より好ましくは10μMから選ばれ、濃度Cは、0.5~2μM、好ましくは0.7~1.5μM、より好ましくは1μMから選ばれる組合せをあげることができる。また、例えばCHIR99021を用いた場合、これに限定されないが、工程(a)、(b)および(c)におけるWntアゴニストの濃度A、BおよびCの組み合わせとして、濃度Aは、6~20μM、好ましくは7~15μM、より好ましくは10μMから選ばれ、濃度Bは、2~6μM、好ましくは2~4.5μM、より好ましくは3μMから選ばれ、濃度Cは、0.5~2μM、好ましくは0.7~1.5μM、より好ましくは1μMから選ばれる組合せをあげることができる。
本発明で用いることができるWntアゴニストは、上記の定義に含まれる限り、天然物または合成物のいずれも含まれ、また、タンパク質、高分子、低分子のいずれであってもよい。本発明において用いることができるWntアゴニストの例としては、これに限定されないが、例えば、好ましくは、GSK-3阻害剤、より好ましくは、CHIR99021、BIO、またはSB415286をあげることができ、特に好ましくは、CHIR99021をあげることができる。各Wntアゴニストの使用濃度は使用目的に合わせて適宜選択できるが、例えば、CHIR99021を用いた場合に得られる効果と同様の効果を発揮できる濃度を選択することができる。
好ましい態様において、工程(b)において用いられるアクチビンは、いずれの起源のアクチビンを用いることができるが好ましくはヒトアクチビンAである。また、培地中の濃度は、分化誘導の効果が得られる限り特に制限がないが、例えば、2.5~40ng/mL、好ましくは7.5~15ng/mLをあげることができる。
また、別の好ましい態様において、工程(b)において用いられるレチノイン酸の培地中の濃度は、分化誘導の効果が得られる限り特に制限がないが、例えば、0.001~1μM、好ましくは0.01~0.3μMをあげることができる。
マウスの多能性幹細胞を用いてネフロン前駆細胞を誘導する場合は、培養は、工程(a)は、例えば、1~4日、好ましくは2日~3日、特に好ましくは2.5日行い、工程(b)は、好ましくは0.5日~2日、特に好ましくは1日行い、工程(c)は、例えば、0.5~3日、好ましくは1日~2.5日、特に好ましくは2日行うことができる。
一方、ヒトの多能性幹細胞を用いてネフロン前駆細胞を誘導する場合は、培養は、工程(a)は、例えば、3~11日、好ましくは4日~10日、特に好ましくは6日行い、工程(b)は、好ましくは1日~3日、特に好ましくは2日行い、工程(c)は、例えば、1~5日、好ましくは2日~4日、特に好ましくは3日行う。
また、共培養後の各構成細胞の分取は、これに限定されないが、例えば、トリプシン等を用いた細胞解離処理後に各細胞特異的な膜たんぱく質(例えば、糸球体上皮細胞であればpodocalyxin、近位尿細管であればCadherin6、遠位尿細管であればEcadherin)を抗体染色し、FACS(フローサイトメーター)を用いて分取することが可能である。
(1)変異マウスの作成
GFPはエクソン2のAgeI部位に挿入し、Osr1のN末端12アミノ酸がGFPにインフレームで結合するようにした。5’Hpa1-AgeI Osr1ゲノム断片(2.8kb)をEGFPに結合し、そしてTakasatoら(Mech Dev 121, 547-557, 2004)の方法に従い、3’BamHI-BamHI断片(5.5kb)を、loxP部位に隣接したNeoおよびタンデムなDTAを含むベクターに挿入した。標的ベクターをE14.1ES細胞にエレクトロポレーションし、次いで、3つの480G418耐性クローンを、BamHIで消化した後、5’プローブを用いたサザンブロット分析により、標的として決定した。正しく標的とされたESクローンを、C57BL/6J雌マウスと交配する生殖系列キメラを作成するために使用した。Neoを、Osr1-GFP変異マウスと遍在的にCreを発現するマウスとを交配させることにより取り除くと、表現型およびEGFP発現パターンは、元の変異マウスのものと同一であった。子孫の遺伝子型分析は、フォワードプライマー、5’-TATGTTGAGGGGGCAGTAGGTTC-3’、2つのリバースプライマー、5’-GTTGGGCAGGTGGTCCGAGGGCA-3’および5’-TAGGTCAGGGTGGTCACGAGGGT-3’を用いたPCRによって行い、野性型アレルのための320塩基対の産物および変異アレルのための420bpの産物を製造した。TnEGFP-CreERT2/+(Acc. No. CDB0604K: http:// www.cdb.riken.jp/arg/mutant%20mice%20list.html)、Wt1tm1(EGFP/cre)Wtpマウス、Six2tm3(EGFP/cre/ERT2)Amcマウス、およびGt(ROSA)26Sortm9(CAG-tdTomato)Hzeマウスは、Jackson Laboratory(米国)から購入した。全ての動物実験は、施設のガイドラインおよび倫理委員会に従って行った。
インビトロコロニー形成アッセイは、Osafuneら(非特許文献4:Development 133, 151-161, 2006)の記載に従って行った。要約すれば、前駆細胞は、FACS Aria II(Becton Dickinson)を用いて選別し、Wnt4を安定に発現するNIH3T3細胞上に低密度(1,250-10,000細胞/ウェル、6ウェルプレート)で播種した。次いで、細胞を、5% ノックアウト血清代替物(Invitrogen)、10μg/mL インスリン、6.7μg/L 亜セレン酸ナトリウム、5.5μg/mL トランスフェリン、1×10-7mol/L デキサメタゾン、10mmol/L ニコチンアミド、2mmol/L L-グルタミン、50μM/L 2-メルカプトエタノール、5mmol/L HEPESおよびペニシリン/ストレプトマイシンを含むDMEM/F12で培養した。
検体は、10%ホルマリンで固定し、パラフィンに包埋して6μmの切片にカットした。免疫染色は、BlueMapまたはDABmapキットおよび自動Discovery System(Roche)を用いて自動的に行うか、または手動で免疫蛍光染色を行った。蛍光免疫組織化学のために、パラフィンで固定した切片を脱パラフィンした。クエン酸緩衝液(pH6.0)中で、5分間、121℃でオートクレーブして脱パラフィンした。室温で1時間、ブロッキング溶液中でインキュベートした後、切片を4℃で一次抗体と共に一晩インキュベートした。次いで、Alexa Fluor 488、561、594、633または647と結合させた二次抗体とインキュベートした。核はDAPI(Roche)でカウンター染色した。凍結切片については、サンプルを4%パラホルムアルデヒドで固定し、最適切削温度(OCT)化合物(Tissue Tek)に包埋し、10μm厚の凍結切片とした。免疫染色のために、OCT化合物をPBSで3回洗浄することにより除去した後、切片をブロッキング溶液中でインキュベートした。以降の手順は、パラフィン切片を染色する場合と同じとした。
E8.5における胚組織培養物については、6-10体節期胚の前体節領域を採取し、T-GFP+細胞をFACSで選別した。選別した細胞を96ウェルの低細胞接着プレートに、凝集体あたり7,000細胞で凝集させて、無血清既知組成培地で培養した。E9.5での胚組織培養では、22から26体節期胚の23体節領域からの尾部領域を回収し、Osr1-GFP+またはWt1-GFP+細胞をFACSで選別した。選別された細胞を、96ウェルの低細胞接着プレート内に凝集体当たり10,000細胞で凝集させて、無血清既知組成培地で培養した
マウスES細胞(Osr1-GFP)を、15%ウシ胎児血清、0.1mMの2-メルカプトエタノール(ナカライテスク)および1,000U/mLの白血病抑制因子(ESGRO)を補充したDMEM(Invitrogen)中にてマウス胚性線維芽細胞上で維持した。EB3-DsRedの細胞は、丹羽仁史博士(理化学研究所CDB)から供与を受けた。EB3-DsRedの細胞は、Usuiら(Am J Pathol 180, 2417-2426, 2012)らの報告に従って維持した。分化を開始する前に、ES細胞を、15%ウシ胎児血清、0.1mM 2-メルカプトエタノール、1,000U/mL 白血病抑制因子、3μM CHIR99021(和光)および1μM PD0325901(和光)を補充したDMEM(Invitrogen)中にて、フィーダー細胞を含まないゼラチンコートディッシュ上で1代継代した。ES細胞の分化は、以下のようにして無血清培地中で行った。ES細胞は、Accutase(ESGRO)で解離し、75%のイスコフ改良ダルベッコ培地(Invitrogen)および25%のハムF12培地(Invitrogen)に、0.5×N2と0.5×B27(レチノイン酸なし)サプリメント(Invitrogen)、0.5×ペニシリン/ストレプトマイシン、0.05%ウシ血清アルブミン、2mM グルタミン(Invitrogen社)、0.5mM アスコルビン酸(シグマ)および4.5×10-4M 1-チオグリセロールを補充した無血清分化培地中で培養した。集めた細胞は、96ウェルの低細胞結合プレートで、凝集体あたり1,000細胞で凝集させ、胚様体(EBs)を形成した。48時間後(day2)、EBsをAccutaseで解離し、0.5ng/mL ヒトアクチビンA(R&D Systems)を加えた無血清分化培地で再凝集させた。24時間後(day3)、培地を、1ng/mL ヒトBmp4(R&D Systems)および10μM CHIR99021を含むBC10培地に切り替えた。36時間後(day4.5)、培地を新しい培地(BC10)に変えた。Day5.5で、培地を10ng/mL アクチビン、3ng/mL Bmp4、3μM CHIR99021および0.1μM レチノイン酸を含むABC3R培地に変更した。day6.5に、培地を、1μM CHIR99021および5ng/mL ヒトFgf9(R&D Systems)を含むC1F培地に変更した。
Kispertら(非特許文献8:Development 125, 4225-4234, 1998)とOsafuneら(非特許文献4:Development 133, 151-161, 2006)の報告に従い、マウス胚後腎間葉細胞または誘導したES細胞凝集体を、10%ウシ胎児血清を含むDMEMを用いて、ポリカーボネートフィルター(0.8μm、ワットマン)の上で、空気-液体界面で、E11.5またはE12.5胚から取った胚脊髄とともに、または3T3Wnt4細胞上で培養した。
胚組織またはES細胞から誘導された細胞凝集体を、5分間、0.25%トリプシンと共にインキュベーションすることによって分離した。正常マウス血清(Thermo Scientific)でブロッキングした後、細胞表面マーカー染色を1%ウシ血清アルブミン、1×HBSSおよび0.035% NaHCO3を含む緩衝液中で行った。データは、ソフトウェアFlowJo(Treestar)で分析した。
用いた抗体は以下のものである:ウサギ抗Pax2(Covance;1:800);フルオレセイン抗LTL(FL-1321;Vector Laboratories;1:100);ニワトリ抗GFP(Abcam;1:1000);ウサギ抗GFP(Invitrogen;1:400);ウサギ抗Itga8(Sigma;1:200);ウサギ抗Pdgfra(Cell Signaling Technology;1:500);マウス抗Pdgfra(Takakuraら、J Histochem Cytochem 45, 883-893, 1997)(1:500);ウサギ抗Wt1(Santa Cruz Biotechnology;1:200);マウス抗Wt1(Dako;1:100);ウサギ抗Six2(Proteintech;1:500);マウス抗Sall1(PPMX Perseus Proteomics;1:200);マウス抗E-cadherin(BD Biosciences;1:800);ウサギ抗Cdh6(Dr. Dressler(Choら、Development 125, 803-812, 1998)から供与;1:400);マウス抗Aqp1(Abcam;1:100);ウサギ抗Podocin(淺沼博士より供与(Lydiaら、 Am J Nephrol 35, 58-68., 2012);1:400);モルモット抗Nephrin(Progen;1:200);ウサギ抗CD31(Abcam;1:25);ラット抗CD34(Abcam;1:100);ウサギ抗DsRed(Clontech;1:100)。
RNAをRNeasy Plus Micro Kit(Qiagen)を用いて単離し、次にランダムプライマーおよびSuperscript III(Invitrogen)を用いて逆転写した。定量的PCRは、Real-Time PCR System(Applied Biosystems)およびThunderbird SYBR qPCR Mix(Toyobo)を用いて行った。すべてのサンプルは、相対標準曲線法を用いてβ-アクチンの発現により標準化した。
標本は、以下の7種類を比較した:E8.5胚のOsr1-GFP陽性および陰性細胞;E9.5胚の尾体幹のOsr1-GFP+/Itga8+/Pdgfra-集団、Osr1-GFP+(但しItga8+/Pdgfra-を除く)集団、およびOsr1-GFP陰性集団;E11.5にて手動操作で分離した後腎間葉のOsr1-GFP+/Itga8+/Pdgfra-集団およびOsr1-GFP+(Itga8+/Pdgfra-を除く)集団。マイクロアレイ分析は、Agilent SurePrint G3マウス遺伝子発現(8×60K)マイクロアレイを用いて行った。データは、GeneSpring GXソフトウェア(アジレント)によって標準化した。マイクロアレイデータは、Biotechnology Information Gene Expression Omnibus(GSE)のためにナショナルセンターに寄託した。
実施例1:コロニー形成前駆細胞を表すOsr1+/Integrina8+/Pdgfra-集団
後腎間葉は、転写因子Six2を発現する間葉を標識する工程を含む細胞運命分析によって示されるように、糸球体の上皮(足細胞を含む)とネフロンの主要部分を構成する細管を生じさせる。発明者らは以前、新規のコロニー形成アッセイを確立することによってネフロン前駆細胞の存在を証明した。Sall1を強発現する分離した後腎間葉系細胞を、Wnt4を安定に発現するフィーダー細胞上に播いた場合、コロニーを形成した単一の細胞群は、糸球体及び腎臓の尿細管マーカーを発現した(非特許文献9:Nishinakamuraら、Development 128, 3105-3115, 2001;非特許文献4:Osafuneら、Development 133, 151-161, 2006)。したがって、Sall1が高くSix2陽性の後腎間葉は、胚腎臓でネフロン前駆体集団を表す。
Osr1は、別の後腎間葉マーカーであり、また、中間中胚葉の最も早いマーカーの1つである。したがって、Osr1は、腎臓発生を通じて腎前駆体集団で連続的に発現される(非特許文献10:Jamesら、Development 133, 2995-3004, 2006;非特許文献1:Mugfordら、Dev Biol 324, 88-98, 2008)。そこで、Osr1-GFPノックインマウスを作成し(図3)、緑色蛍光タンパク質(GFP)が、E8.5-E9.5の中間中胚葉で、そして、E11.5-E15.5の後腎間葉で発現することを確認した(図4A~C)。
次に、ネフロン前駆細胞マーカーの発現、および初期段階のOsr1-GFP陽性細胞のコロニー形成能力を検討した。図5および表1に示すように、E8.5では、Itga8とGFPの重複は検出されず、GFP+集団によるコロニー形成もなかったが、E9.5では、GFP+集団によるコロニー形成が検出された(0.037±0.013%)。
また、Itga8+/Pdafra-であるGFP+領域を見つけ(図5)、そして、Osr1+/Itga8+/Pdgfra-集団を選別することにより、コロニー形成細胞を濃縮した(1.10±0.26%、図6、表2)。しかし濃縮後でさえ、コロニー形成頻度は、E10.5およびE11.5の後腎領域からのOsr1+/Itga8/Pdgfra-集団のそれより有意に低かった(それぞれ、30.9±1.5%及び50.9±5.2%、表2)。これとは対照的に、E10.5の中腎領域からのOsr1+/Itga8+/Pdgfra-集団のコロニー形成頻度(1.47±0.20%、表2)は、E9.5のそれと同じくらい低かった。後腎の前方に位置している中腎は、後腎より早く発展し、はるかに少ないネフロンを形成するので、E9.5でのコロニー形成細胞は、中腎ネフロン前駆細胞を表すかもしれないという仮説を立てた。
Osr1+/Itga8+/Pdgfra-であるコロニー形成が推定されるE9.5の中腎前駆細胞と、E10.5~11.5の後腎ネフロン前駆細胞を用いて、マイクロアレイと定量PCR解析を行った。結果を図13と図14に示す。両方のタイプの前駆細胞が、例えば、Osr1、Wt1、Pax2およびSix2のような共通の転写因子、ならびにGdnf(腎臓発生に必須のサイトカイン)を発現している一方、後腎前駆細胞はHoxa10、Hoxa11およびHoxd12を含む尾部Hox遺伝子をより多く発現していた。E9.0前後で胚の後端で発現され始めるHox11ファミリー遺伝子は、中間中胚葉の前後軸に沿った後腎領域を決定づけることによって後腎の発達に不可欠であると報告されている。さらに、細胞運命マッピング研究により、E9.5のOsr1+中間中胚葉が後腎間葉に寄与していることが示されている。それゆえ、E9.5で後方に位置していた非コロニー形成Osr1+/尾部Hox+である中間中胚葉は、後腎ネフロン前駆細胞の前駆体集団となり得るという仮説を立てた(図15、図11および図13)。特に、E9.5の尾部中間中胚葉におけるPax2、Six2およびGdnfの発現レベルは、依然としてE10.5での尾部後腎前駆細胞に比べてはるかに低く、このことは、それらが異なっていることを示している(図13)。
したがって、1μM CHIRおよびFgf9の組み合わせ(C1F)は、尾部中間中胚葉から後腎ネフロン前駆細胞への誘導に最適であった。これらの観察結果は、後腎間葉の形成と維持のためのFfg受容体とFgf9/Fgf20がそれぞれ要求されることを示している先行文献と一致する(非特許文献11:Barakら、Dev Cell 22, 1191-1207., 2012、および非特許文献12:Poladiaら、Dev Biol 291, 325-339, 2006)。
次に、E9.5尾部中間中胚葉の方法で、早い段階の中胚葉を後腎間葉に分化させるインビトロでの方法で調べた。一つの報告では、中間中胚葉由来の後腎間葉と尿管芽の両方が、胎生(E)8.5前後で現れ、転写因子Osr1を発現することが示されている(非特許文献1:Mugfordら、Dev Biol 324, 88-98, 2008)。他のいくつかの報告では、尿管芽は、前方中間中胚葉に由来し、そしてその原基であるウォルフ管は、前方から後方に向けて伸長することが、ニワトリ胚を直接標識することにより示されている(非特許文献13:Atsutaら、Dev Growth Differ 55, 579-590, 2013、非特許文献14:Attiaら、Development 139, 4143-4151, 2012、非特許文献15:Obara-Ishiharaら、Development 126, 1103-1108, 1999、非特許文献16:Saxen, Cell 131, 861-872, 1987)。マウス胚では、E8.5のPax2/8-陽性前中間中胚葉(前腎原基と呼ばれる)は、同等の集団と推定され、Osr1-陽性領域に含まれる。従って、最初に、選別されたE8.5のOsr1-GFP+細胞を用いて増殖因子の多くの組み合わせの効果を検討した。しかし、コロニー形成前駆細胞を誘導することができなかった。
以上をまとめると、図21に示されるように、後腎間葉の前駆体は、E8.5後の原腸形成の段階まで、T陽性状態に維持され後方化される。おそらくこれは、部分的には、尾体幹の供給源として最近認識された”体軸幹細胞(axial progenitor)”に対応すると考えられる。これらのデータはまた、後方に位置する後腎間葉組織および前方に位置する中胚葉組織、例えば心臓、の間の発生過程の違いを明らかにする。なお、これらの前方に位置する中胚葉組織は、分化初期における短期間のT陽性状態を経て多能性幹細胞からの誘導に成功している(非特許文献18:Burridge、Cell Stem Cell 10, 16-28, 2012)。
系統トレース実験に基づき、原料として選別したE8.5のT+尾部中胚葉を用いて、後腎ネフロン前駆細胞を誘導した。結果を図18に示す。選別した細胞は、再凝集させて凝集体を形成させて種々の成長因子で処理し、次いで上記のようにC1Fで処理した(図22C、工程3)。後方中間中胚葉は、Osr1、Wt1および尾部Hox遺伝子の発現によって識別されるので、まずこれらの遺伝子の発現に影響を与える可能性がある成長因子に注目した。BmpおよびWntシグナル伝達の相乗効果は、マウス胚性幹(ES)細胞分化における尾部Hox遺伝子の発現で報告されており、レチノイン酸シグナル伝達は、ゼブラフィッシュ発達におけるWt1同族の発現に重要であると報告されている。レチノイン酸、BmpおよびWntアゴニストの同時導入は、胚後腎間葉で観察されたレベルまで尾部Hox遺伝子を増加させなかった(図22DとE)ので、”後方化期”を追加した。すなわち、カノニカルなWntシグナルが体幹の伸長に重要であると報告されているので、Bmp4と組み合わせて、Wntアゴニストを高濃度(10μM CHIR)で添加した。この処理(BC10、ステップ1)は劇的に尾部Hox遺伝子の発現を増加させ、そして、腎臓遺伝子の発現レベルは胚後腎間葉に比べてまだ低かったが、誘導された細胞からのコロニー形成を検出することができた(図22DとG)。次に、様々な条件を試み、アクチビンとレチノイン酸の組み合わせを、Bmp4および3μM CHIRと一緒に用いること(ABC3R、ステップ2)が、腎臓遺伝子の発現を実質的に増加させることを見いだした(図22F)。この最適化された3段階の条件(BC10とそれに続くABC3RおよびC1F)で、細胞は、アクチビンまたはレチノイン酸のいずれかの単独での添加に比べて、多数のコロニー(22.7±3.66%、n=4)を形成した(図22G)。最初の後方化工程において、標準的なWntアゴニストの濃度は極めて重要であり、レチノイン酸の添加は完全にコロニー形成前駆細胞誘導を阻害した(図23A)。第二工程において、アクチビン、レチノイン酸、Bmp、WntまたはFgfシグナリングのいずれかの阻害は、コロニー形成を減少させ(図23B)、このことは、これらのシグナルのすべてが必須であることを示唆している。
ES細胞からの後腎ネフロン前駆細胞の誘導を行った。ES細胞からの後腎間充織の誘導の全体の工程の概要を図1に示し、各段階におけるシグネチャー遺伝子の発現を図25に示す。中間中胚葉と後腎ネフロン前駆細胞の誘導を監視するために、上記したOsr1-GFPマウスから作成したOsr1-GFP ES細胞株を用いて、2日間、因子なしの無血清培地で培養して胚様体(EBs)を作成した。EBsを用いて、後腎ネフロン前駆細胞の作成を行った。次の24時間、低濃度のアクチビンを添加して、胚性外胚葉マーカーFgf5の一過性の発現を誘導した。EBsはその後、Bmp4と高濃度のCHIRで処理した(工程2)。4.5日では、尾部新生前駆体を表すT、ならびにCdx2およびTbx6の発現がアップレギュレートされた。その後、上記の胚後部中胚葉のためのプロトコルを完全に再現できた(図22C、および図1の工程3~5)。day8.5に収穫した誘導されたEBsは、胚後腎間葉に匹敵するレベルで、後腎ネフロン前駆細胞に対する複数のシグネチャー遺伝子を発現していた(図25)。免疫染色の結果は、多くの細胞が、Osr1、Wt1、Pax2、Sall1およびSix2を含む典型的な腎性転写因子を共発現していることを示した(図26)。さらに、FACS分析の結果は、細胞の約90%近くがOsr1-GFP陽性であり、Osr1+細胞の中でItga8+/Pdgfra-前駆細胞は約65%を構成することを示した(図27)。これらの誘導された前駆細胞は、堅牢なコロニー形成を(21.3±1.69%、n=8)を示した。このことは、ES細胞から後腎ネフロン前駆細胞を生成することに成功したことを示している。
E11.5胚からの後腎間葉は、気液界面にて胚脊髄またはWnt4発現細胞と共培養したとき、間葉上皮転換し、糸球体および尿細管を形成することが十分に確立されている(非特許文献8:Kispertら、Development 125, 4225-4234, 1998)。E11.5胚を用いて作成した糸球体および尿細管を図28Aに示す。そこで、実施例6で誘導したEBsを同様の方法で培養したところ、しっかりとした尿細管形成が確認された。結果を図28CおよびDに示す。特に、day6で収穫したEBsの組織学的検査では、いずれの条件でも多くの管形成が認められ(図28CとD)、これはE-カドヘリン染色によって確認された(図28E)。管のほとんどは、Pax2とSall1陽性であり、腎尿細管であることを示している(図28E~H、L~P)。いくつかの尿細管は、近位尿細管のマーカーである、例えば、LTLまたはカドヘリン6、アクアポリン1、Jagged1、Megalinなどを発現していた(図28F、G、L~N)。他の尿細管は、遠位尿細管の形成を示すE-カドヘリン、Brn1,NCCを発現していた(図28H、O、P)。さらに素晴らしいことに、多数の糸球体様構造が観察された(図28Cおよび28I~28K)。これらの構造は、核に典型的な糸球体上皮細胞マーカーであるWt1を発現する細胞のクラスターで、ネフリンやポドシンなどの足突起タンパク質も発現していた。これらの構造は、胚後腎間葉のものと区別できなかった(図28BおよびD、n=6)。
上記のマウスES細胞に用いたプロトコルをヒトiPS細胞に適用し、インビトロで、iPS細胞を後腎ネフロン前駆細胞に向かって分化させた。iPS細胞からの後腎間葉の誘導の全体の工程の概要を図2に示した。以前の報告では、ヒト多能性幹細胞において、中胚葉系譜の細胞の初期誘導のためには、Bmp、Fgfおよびアクチビンのシグナルが重要であることが示されている(非特許文献19:Bernardら、Cell Stem Cell 9, 144-155, 2011; 非特許文献20:Kattmanら、Cell stem cell 8, 228-240, 2011; 非特許文献21:Yuら、Cell Stem Cell 8, 326-334, 2011)。したがって、ヒトiPS細胞凝集体を、最初の24時間Bmpで処理し、次いで、2日間アクチビンおよびFgfで処理した。誘導された中胚葉細胞をさらに、マウスES細胞の誘導と同様に、高濃度のWntアゴニスト(CHIR 10μM)およびBmpの存在下で、後方化しつつ未熟な中胚葉状態に維持した。ヒト胚における体幹伸長のための生理的期間を考え、これらの培養条件下で6日間EBsを培養した。その後、単に培養期間を調整することにより、マウスES細胞の分化のためのプロトコルを完全に再現した。14日目に採取した誘導EBsは、後腎ネフロン前駆細胞の複数のシグネチャー遺伝子を発現していた(図30)。免疫染色の結果、多くの細胞が、Wt1、Pax2、Sall1およびSix2を含む代表的な腎性転写因子を共発現していることが明らかなった(図31)。これらの誘導前駆細胞は、マウス胚脊髄と共培養すると、堅牢な尿細管形成および糸球体上皮細胞の形成を示した(図32DとE、n=6)。10日目での免疫組織化学的検査の結果は、十分に特定されたネフロン成分の形成を明らかにした。これらの構造は、Wt1/ネフリン(Nephrin)陽性糸球体(図32K、32J)、カドヘリン6(cdh6)陽性近位尿細管(図32H)およびE-カドヘリン陽性遠位尿細管(図32I)で構成され、それらの全てがその順で連結されており、それによってヒト胎児腎臓形成を再現していた。
以上のように、インビボでの発生過程を反復することにより、インビトロで、マウスおよびヒト多能性幹細胞の両方から真正な後腎ネフロン前駆体および三次元腎臓構造の誘導に成功した(図33)。
Claims (31)
- 哺乳動物由来の多能性幹細胞から後腎ネフロン前駆細胞を分化誘導する方法であって、該方法が、以下の3つの工程を、
(a)多能性幹細胞から誘導された胚様体を、Bmpおよび高濃度(濃度A)のWntアゴニストを含有する培地で培養する工程、
(b)前記胚様体を、Bmpおよび中濃度(濃度B)のWntアゴニストを含有する培地で培養する工程、および
(c)前記胚様体を、Fgfおよび低濃度(濃度C)のWntアゴニストを含有する培地で培養する工程、
をその順で含むことを特徴とする分化誘導方法
(ここで、Wntアゴニストの濃度は、濃度A>濃度B>濃度Cであって、濃度Aは濃度Cの少なくとも5倍である。)。 - 前記工程(a)、(b)および(c)におけるWntアゴニストの濃度は、濃度Aは濃度Bの少なくとも3倍であり、かつ濃度Bは濃度Cの少なくとも3倍である、請求項1に記載の分化誘導方法。
- 前記工程(b)において、培地がさらにアクチビンを含む、請求項1または2に記載の分化誘導方法。
- 前記工程(b)において、培地がさらにレチノイン酸を含む、請求項3に記載の分化誘導方法。
- 前記WntアゴニストがGSK-3阻害剤である(ただし、各工程のWntアゴニストは同じであっても異なってもよい)、請求項1~4のいずれかひとつに記載の分化誘導方法。
- 前記Wntアゴニストが、CHIR99021、BIO、およびSB415286からなる群より選ばれる(ただし、各工程のWntアゴニストは同じであっても異なってもよい)、請求項5に記載の分化誘導方法。
- 前記Bmpが、Bmp2、Bmp4およびBmp7からなる群より選ばれる、かつ、前記Fgfが、Fgf2、Fgf9およびFgf20からなる群より選ばれる、請求項1~6のいずれかに記載の分化誘導方法。
- 前記BmpがBmp4であり、かつ前記FgfがFgf9である、請求項1~7のいずれか一つに記載の分化誘導方法。
- 前記工程(a)、(b)および(c)におけるWntアゴニストが、CHIR99021であり、かつ、前記濃度Aが7.5μM~15μM、前記濃度Cが0.5μM~2.0μMである、請求項1~8のいずれか一つに記載の分化誘導方法。
- 前記工程(a)および(b)におけるBmpがBmp4であり、工程(a)における濃度が0.1ng/ml~3ng/mlであり、工程(b)における濃度が1ng/ml~10ng/mlである、請求項9に記載の分化誘導方法。
- 前記工程(b)において、アクチビンを2.5~40ng/mLの濃度で含む請求項10に記載の分化誘導方法。
- 前記工程(a)、(b)および(c)が、連続した工程である請求項1~11のいずれか一つに記載の分化誘導方法。
- 前記工程(c)において、培地が、Bmp、アクチビンまたはレチノイン酸のいずれも含まない培地である、請求項1~12のいずれか一つに記載の分化誘導方法。
- 前記多能性幹細胞がマウスES細胞またはiPS細胞、またはヒトES細胞またはiPS細胞である、請求項1~13のいずれか一つに記載の分化誘導方法。
- 前記多能性幹細胞がヒトiPS細胞である、請求項14に記載の分化誘導方法。
- 前記多能性幹細胞がマウスES細胞またはiPS細胞であり、かつ工程(a)が、少なくとも1日以上4日以内培養する工程である請求項14に記載の分化誘導方法。
- 前記多能性幹細胞がヒトES細胞またはiPS細胞であり、かつ工程(a)が、少なくとも3日以上11日以内培養する工程である請求項14に記載の分化誘導方法。
- 転写因子である、Osr1、Wt1、Pax2、Six2、Hoxa10、Hoxa11の全てを発現する細胞集団であることを特徴とする哺乳動物由来の多能性幹細胞から分化誘導されたネフロン前駆細胞。
- 前記多能性幹細胞が、マウスES細胞またはiPS細胞、またはヒトES細胞またはiPS細胞である、請求項18に記載のネフロン前駆細胞。
- 請求項1~17のいずれか一つに記載の分化誘導方法により誘導されたネフロン前駆細胞。
- 請求項18~20のいずれか一つに記載のネフロン前駆細胞を用いて、糸球体および尿細管を有する三次元腎臓構造を作成する方法。
- 前記方法が、ネフロン前駆細胞を気液界面にて胚脊髄またはWnt4発現細胞と共培養ことを含む、請求項21に記載の三次元腎臓を作成する方法。
- 請求項21または22に記載の方法により形成された、糸球体および尿管を有する三次元腎臓構造。
- 請求項18~20のいずれか一つに記載のネフロン前駆細胞から分化誘導されたCadherin6、Megalin、およびLTLを発現する細胞集団であることを特徴とする近位尿細管細胞。
- 前記分化誘導が、ネフロン前駆細胞を胎児脊髄またはWnt4発現細胞と共培養することにより行われる、請求項24に記載の近位尿細管細胞。
- 請求項18~20のいずれか一つに記載のネフロン前駆細胞から分化誘導された、E-cadherin、Brn1、およびNCCを発現する細胞集団であることを特徴とする遠位尿細管細胞。
- 前記分化誘導が、ネフロン前駆細胞を胎児脊髄またはWnt4発現細胞と共培養することにより行われる、請求項26に記載の遠位尿細管細胞。
- 請求項18~20のいずれか一つに記載のネフロン前駆細胞から分化誘導された、Wt1、Nephrin、およびPodocinを発現する細胞集団であることを特徴とする糸球体上皮細胞。
- 前記分化誘導が、ネフロン前駆細胞を胎児脊髄またはWnt4発現細胞と共培養することにより行われる、請求項28に記載の糸球体上皮細胞。
- 多能性幹細胞からネフロン前駆細胞を誘導するための分化誘導培地キットであって、該キットは、(i)分化誘導培地、および(ii)Wntアゴニストを含み、使用時において、前記Wntアゴニストを段階的濃度で含む少なくとも3つの分化誘導培地が調整される、ここで、前記段階的濃度は、少なくとも濃度A、BおよびCからなり、濃度A>濃度B>濃度Cであって、濃度Aは濃度Cの少なくとも5倍である、
ことを特徴とする培地キット、培地キット。 - 前記Wntアゴニストの段階的濃度は、濃度Aは濃度Bの少なくとも3倍であり、かつ濃度Bは濃度Cの少なくとも3倍であるように分化誘導培地が調整される、請求項30に記載の培地キット。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP14853879.6A EP3059308B1 (en) | 2013-10-18 | 2014-10-16 | Method of inducing kidney from pluripotent stem cells |
JP2015542663A JP6455729B2 (ja) | 2013-10-18 | 2014-10-16 | 多能性幹細胞からの腎臓誘導法 |
US15/026,462 US10072249B2 (en) | 2013-10-18 | 2014-10-16 | Method of inducing kidney from pluripotent stem cells |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2013217029 | 2013-10-18 | ||
JP2013-217029 | 2013-10-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2015056756A1 true WO2015056756A1 (ja) | 2015-04-23 |
Family
ID=52828197
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2014/077601 WO2015056756A1 (ja) | 2013-10-18 | 2014-10-16 | 多能性幹細胞からの腎臓誘導法 |
Country Status (4)
Country | Link |
---|---|
US (1) | US10072249B2 (ja) |
EP (1) | EP3059308B1 (ja) |
JP (1) | JP6455729B2 (ja) |
WO (1) | WO2015056756A1 (ja) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017010448A1 (ja) * | 2015-07-11 | 2017-01-19 | 国立大学法人熊本大学 | ネフロン形成能を有するネフロン前駆細胞の増幅培養方法 |
WO2017041041A1 (en) | 2015-09-03 | 2017-03-09 | The Brigham And Women's Hospital, Inc. | Three-dimensional differentiation of epiblast spheroids to kidney organoids models stage-specific epithelial physiology, morphogenesis, and disease |
JP2018527007A (ja) * | 2015-09-17 | 2018-09-20 | ザ ブリガム アンド ウィメンズ ホスピタル インコーポレイテッドThe Brigham and Women’s Hospital, Inc. | ヒト多能性幹細胞からネフロンを生成する方法 |
JP2018183137A (ja) * | 2017-04-25 | 2018-11-22 | 国立大学法人 熊本大学 | 多能性幹細胞から樹状分岐した集合管を伴う腎臓構造を作製する方法 |
WO2018216743A1 (ja) * | 2017-05-25 | 2018-11-29 | 国立大学法人京都大学 | 中間中胚葉細胞から腎前駆細胞への分化誘導方法、および多能性幹細胞から腎前駆細胞への分化誘導方法 |
JP2019506899A (ja) * | 2016-01-25 | 2019-03-14 | アルベルト−ルートヴィヒ−ウニベルシタット フライブルクAlbert−Ludwigs−Universitaet Freiburg | 分化細胞からの腎細胞の作製方法 |
CN110468095A (zh) * | 2019-09-05 | 2019-11-19 | 安徽中盛溯源生物科技有限公司 | 一种肾上皮细胞及其制备方法和应用 |
JPWO2019230737A1 (ja) * | 2018-05-29 | 2021-08-12 | 国立大学法人 熊本大学 | 糸球体ポドサイトの誘導方法、及び該誘導方法を用いた多能性幹細胞からのポドサイトの製造方法 |
WO2023077148A1 (en) | 2021-11-01 | 2023-05-04 | Tome Biosciences, Inc. | Single construct platform for simultaneous delivery of gene editing machinery and nucleic acid cargo |
WO2024020587A2 (en) | 2022-07-22 | 2024-01-25 | Tome Biosciences, Inc. | Pleiopluripotent stem cell programmable gene insertion |
WO2024138194A1 (en) | 2022-12-22 | 2024-06-27 | Tome Biosciences, Inc. | Platforms, compositions, and methods for in vivo programmable gene insertion |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US12024721B2 (en) * | 2017-08-18 | 2024-07-02 | Wisconsin Alumni Research Foundation | Method of differentiating human pluripotent stem cells to podocytes |
KR102150489B1 (ko) * | 2019-04-09 | 2020-09-01 | 고려대학교 산학협력단 | 소변세포로부터 신장전구세포로의 직접 역분화를 유도하는 방법 및 이의 방법으로 역분화된 신장전구세포를 포함하는 신장세포 손상 질환 예방 또는 치료용 약학 조성물 |
CN114891737B (zh) * | 2022-04-25 | 2023-05-12 | 中山大学 | 一种多能干细胞来源肾脏间充质干细胞的制备方法及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012011610A1 (en) | 2010-07-21 | 2012-01-26 | Kyoto University | Method for inducing differentiation of human pluripotent stem cell into intermediate mesoderm cell |
WO2013094771A1 (en) * | 2011-12-19 | 2013-06-27 | Kyoto University | Method for inducing differentiation of human pluripotent stem cells into intermediate mesoderm cells |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070031966A1 (en) | 2005-07-18 | 2007-02-08 | Regents Of The University Of Michigan | Renal progenitor cells from embryonic stem cells |
WO2012013969A1 (en) | 2010-07-26 | 2012-02-02 | The University Of Manchester | Targeted differentiation of stem cells |
-
2014
- 2014-10-16 US US15/026,462 patent/US10072249B2/en active Active
- 2014-10-16 WO PCT/JP2014/077601 patent/WO2015056756A1/ja active Application Filing
- 2014-10-16 EP EP14853879.6A patent/EP3059308B1/en active Active
- 2014-10-16 JP JP2015542663A patent/JP6455729B2/ja active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012011610A1 (en) | 2010-07-21 | 2012-01-26 | Kyoto University | Method for inducing differentiation of human pluripotent stem cell into intermediate mesoderm cell |
JP2013530680A (ja) * | 2010-07-21 | 2013-08-01 | 国立大学法人京都大学 | ヒト多能性幹細胞から中間中胚葉細胞への分化誘導方法 |
WO2013094771A1 (en) * | 2011-12-19 | 2013-06-27 | Kyoto University | Method for inducing differentiation of human pluripotent stem cells into intermediate mesoderm cells |
Non-Patent Citations (41)
Title |
---|
ASAYAMA.M.: "EFFECTS OF AN HERG ACTIVATOR, ICA-105574, ON ELECTROPHYSIOLOGICAL PROPERTIES OF CANINE HEARTS", J PHARMACOL SCI, vol. 121, no. SUPPLE, 15 March 2013 (2013-03-15), pages S1C - 6-4, XP055334169 * |
ATSUTA ET AL., DEV GROWTH DIFFER, vol. 55, 2013, pages 579 - 590 |
ATTIA ET AL., DEVELOPMENT, vol. 139, 2012, pages 4143 - 4151 |
BARAK ET AL., DEV CELL, vol. 22, 2012, pages 1191 - 1207 |
BERNARD ET AL., CELL STEM CELL, vol. 9, 2011, pages 144 - 155 |
BURRIDGE ET AL., CELL STEM CELL, vol. 10, 2012, pages 16 - 28 |
BURRIDGE, CELL STEM CELL, vol. 10, 2012, pages 16 - 28 |
CHO ET AL., DEVELOPMENT, vol. 125, 1998, pages 803 - 812 |
DAIWA SHOKEN HEALTH ZAIDAN NO JOSEI NI YORU ... KENKYU GYOSEKISHU, 1 March 2013 (2013-03-01), pages 37 - 42, XP008183599 * |
HERRMANN ET AL., NATURE, vol. 343, 1990, pages 617 - 622 |
INTERNATIONAL SOCIETY FOR STEM CELL RESEARCH POSTER ABSTRACTS, vol. 11TH, 12 June 2013 (2013-06-12), pages F-2184, XP008185221, Retrieved from the Internet <URL:http://www.isscr.org/docs/default-source/am2013-support-documents/isscr2013-poster-abstracts.pdf> * |
JAMES ET AL., DEVELOPMENT, vol. 133, 2006, pages 2995 - 3004 |
JAPANESE JOURNAL OF PEDIATRIC NEPHROLOGY, vol. 26, no. 1, 15 April 2013 (2013-04-15), pages 64 - 69, XP008181646 * |
KATTMAN ET AL., CELL STEM CELL, vol. 8, 2011, pages 228 - 240 |
KISPERT ET AL., DEVELOPMENT, vol. 125, 1998, pages 4225 - 234 |
KISPERT ET AL., DEVELOPMENT, vol. 125, 1998, pages 4225 - 4234 |
KORINEK ET AL., SCIENCE, vol. 275, 1997, pages 1784 - 1787 |
LYDIA ET AL., AM J NEPHROL, vol. 35, 2012, pages 58 - 68 |
MUGFORD ET AL., DEV BIOL, vol. 319, 2008, pages 396 - 405 |
MUGFORD ET AL., DEV BIOL, vol. 324, 2008, pages 88 - 98 |
NAIBUNPITSU.TONYOBYO.TAISHA NAIKA, vol. 35, no. 2, 2012, pages 125 - 129, XP008183420 * |
NATURE COMMUNICATIONS, vol. 4, 22 January 2013 (2013-01-22), pages 1367, XP055184925 * |
NEPHROLOGY FRONTIER, vol. 10, no. 3, 2011, pages 238 - 241, XP008184633 * |
NISHIKAWA.M.: "STEPWISE RENAL LINEAGE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS TRACING IN VIVO DEVELOPMENT", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 417, 2012, pages 897 - 902, XP028441673 * |
NISHINAKAMURA ET AL., DEVELOPMENT, vol. 128, 2001, pages 3105 - 3115 |
OBARA-ISHIHARA ET AL., DEVELOPMENT, vol. 126, 1999, pages 1103 - 1108 |
OSAFUNE ET AL., DEVELOPMENT, vol. 133, 2006, pages 151 - 161 |
OSAFUNE, NISHINAKAMURA ET AL., DEVELOPMENT, vol. 133, 2006, pages 151 - 161 |
OSAFUNE.K.: "IDENTIFICATION OF MULTIPOTENT PROGENITORS IN THE EMBRYONIC MOUSE KIDNEY BY A NOVEL COLONY-FORMING ASSAY", DEVELOPMENT, vol. 133, 2006, pages 151 - 161, XP002581530 * |
PLOS ONE, vol. 9, no. 1, January 2014 (2014-01-01), pages E84881, XP055184928 * |
POLADIA ET AL., DEV BIOL, vol. 291, 2006, pages 325 - 339 |
SAXEN, CELL, vol. 131, 1987, pages 861 - 872 |
SEITAI NO KAGAKU, vol. 65, no. 3, 15 June 2014 (2014-06-15), pages 244 - 248, XP008183419 * |
TAGUCHI.A.: "REDEFINING THE IN VIVO ORIGIN OF METANEPHRIC EPHRON PROGENITORS ENABLES GENERATION OF COMPLEX KIDNEY STRUCTURES FROM PLURIPOTENT STEM CELLS", CELL STEM CELL, vol. 14, 2 January 2014 (2014-01-02), pages 53 - 67, XP055184270 * |
TAKAKURA ET AL., J. HISTOCHEM CYTOCHEM, vol. 45, 1997, pages 883 - 893 |
TAKEMOTO ET AL., NATURE, vol. 470, 2011, pages 394 - 398 |
TZOUANACOU ET AL., DEV CELL, vol. 17, 2009, pages 365 - 376 |
USUI ET AL., AM. J. PATHOL., vol. 180, 2012, pages 2417 - 2426 |
WELLIK ET AL., GENES DEV, vol. 16, 2002, pages 1423 - 1432 |
WILSON ET AL., DEVELOPMENT, vol. 136, 2009, pages 1591 - 1604 |
YU ET AL., CELL STEM CELL, vol. 8, 2011, pages 326 - 334 |
Cited By (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPWO2017010448A1 (ja) * | 2015-07-11 | 2018-05-24 | 国立大学法人 熊本大学 | ネフロン形成能を有するネフロン前駆細胞の増幅培養方法 |
WO2017010448A1 (ja) * | 2015-07-11 | 2017-01-19 | 国立大学法人熊本大学 | ネフロン形成能を有するネフロン前駆細胞の増幅培養方法 |
AU2016318114B2 (en) * | 2015-09-03 | 2023-04-20 | The Brigham And Women's Hospital, Inc. | Three-dimensional differentiation of epiblast spheroids to kidney organoids models stage-specific epithelial physiology, morphogenesis, and disease |
EP3344264A4 (en) * | 2015-09-03 | 2019-02-20 | The Brigham and Women's Hospital, Inc. | THREE-DIMENSIONAL DIFFERENTIATION OF EPIBLASTIC SPHERES OF NIERENORGANOID MODEL STADIUM-SPECIFIC EPITHELEPHYSIOLOGY, MORPHOGENESIS AND DISEASE |
WO2017041041A1 (en) | 2015-09-03 | 2017-03-09 | The Brigham And Women's Hospital, Inc. | Three-dimensional differentiation of epiblast spheroids to kidney organoids models stage-specific epithelial physiology, morphogenesis, and disease |
US10815460B2 (en) | 2015-09-03 | 2020-10-27 | The Brigham And Women's Hospital, Inc. | Three-dimensional differentiation of epiblast spheroids to kidney organoids models stage-specific epithelial physiology, morphogenesis, and disease |
JP2018527007A (ja) * | 2015-09-17 | 2018-09-20 | ザ ブリガム アンド ウィメンズ ホスピタル インコーポレイテッドThe Brigham and Women’s Hospital, Inc. | ヒト多能性幹細胞からネフロンを生成する方法 |
US11046932B2 (en) | 2016-01-25 | 2021-06-29 | Albert-Ludwigs-Universitaet Freiburg | Method of producing renal cells from differentiated cells |
JP2019506899A (ja) * | 2016-01-25 | 2019-03-14 | アルベルト−ルートヴィヒ−ウニベルシタット フライブルクAlbert−Ludwigs−Universitaet Freiburg | 分化細胞からの腎細胞の作製方法 |
JP2018183137A (ja) * | 2017-04-25 | 2018-11-22 | 国立大学法人 熊本大学 | 多能性幹細胞から樹状分岐した集合管を伴う腎臓構造を作製する方法 |
JP7274683B2 (ja) | 2017-04-25 | 2023-05-17 | 国立大学法人 熊本大学 | 多能性幹細胞から樹状分岐した集合管を伴う腎臓構造を作製する方法 |
US11821007B2 (en) | 2017-05-25 | 2023-11-21 | Kyoto University | Method for inducing differentiation of intermediate mesodermal cell to renal progenitor cell, and method for inducing differentiation of pluripotent stem cell to renal progenitor cell |
JP7161775B2 (ja) | 2017-05-25 | 2022-10-27 | 国立大学法人京都大学 | 中間中胚葉細胞から腎前駆細胞への分化誘導方法、および多能性幹細胞から腎前駆細胞への分化誘導方法 |
JP2022188229A (ja) * | 2017-05-25 | 2022-12-20 | 国立大学法人京都大学 | 中間中胚葉細胞から腎前駆細胞への分化誘導方法、および多能性幹細胞から腎前駆細胞への分化誘導方法 |
JPWO2018216743A1 (ja) * | 2017-05-25 | 2020-03-26 | 国立大学法人京都大学 | 中間中胚葉細胞から腎前駆細胞への分化誘導方法、および多能性幹細胞から腎前駆細胞への分化誘導方法 |
WO2018216743A1 (ja) * | 2017-05-25 | 2018-11-29 | 国立大学法人京都大学 | 中間中胚葉細胞から腎前駆細胞への分化誘導方法、および多能性幹細胞から腎前駆細胞への分化誘導方法 |
JP7458012B2 (ja) | 2017-05-25 | 2024-03-29 | 国立大学法人京都大学 | 中間中胚葉細胞から腎前駆細胞への分化誘導方法、および多能性幹細胞から腎前駆細胞への分化誘導方法 |
JPWO2019230737A1 (ja) * | 2018-05-29 | 2021-08-12 | 国立大学法人 熊本大学 | 糸球体ポドサイトの誘導方法、及び該誘導方法を用いた多能性幹細胞からのポドサイトの製造方法 |
JP7421049B2 (ja) | 2018-05-29 | 2024-01-24 | 国立大学法人 熊本大学 | 糸球体ポドサイトの誘導方法、及び該誘導方法を用いた多能性幹細胞からのポドサイトの製造方法 |
CN110468095A (zh) * | 2019-09-05 | 2019-11-19 | 安徽中盛溯源生物科技有限公司 | 一种肾上皮细胞及其制备方法和应用 |
WO2023077148A1 (en) | 2021-11-01 | 2023-05-04 | Tome Biosciences, Inc. | Single construct platform for simultaneous delivery of gene editing machinery and nucleic acid cargo |
WO2024020587A2 (en) | 2022-07-22 | 2024-01-25 | Tome Biosciences, Inc. | Pleiopluripotent stem cell programmable gene insertion |
WO2024138194A1 (en) | 2022-12-22 | 2024-06-27 | Tome Biosciences, Inc. | Platforms, compositions, and methods for in vivo programmable gene insertion |
Also Published As
Publication number | Publication date |
---|---|
US10072249B2 (en) | 2018-09-11 |
EP3059308B1 (en) | 2020-03-18 |
JPWO2015056756A1 (ja) | 2017-03-09 |
US20160304838A1 (en) | 2016-10-20 |
EP3059308A1 (en) | 2016-08-24 |
JP6455729B2 (ja) | 2019-01-23 |
EP3059308A4 (en) | 2017-03-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6455729B2 (ja) | 多能性幹細胞からの腎臓誘導法 | |
US20220025321A1 (en) | Methods for reprograming non-pluripotent cells into pluripotent stem cells | |
JP6931635B2 (ja) | 単離ヒト肺前駆細胞およびその使用 | |
JP7458012B2 (ja) | 中間中胚葉細胞から腎前駆細胞への分化誘導方法、および多能性幹細胞から腎前駆細胞への分化誘導方法 | |
JP6937810B2 (ja) | 腎臓前駆細胞 | |
CN107208051B (zh) | 形成类肾体的多能干细胞的分化 | |
EP2898063B1 (en) | In vitro pancreatic differentiation of pluripotent mammalian cells | |
JP6450311B2 (ja) | 腎前駆細胞の製造方法及び腎前駆細胞を含む医薬 | |
KR20160029115A (ko) | 비-다능성 세포를 다능성 줄기 세포가 되도록 재프로그래밍하기 위한 조성물 및 방법 | |
US11066650B2 (en) | Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same | |
WO2021241621A1 (ja) | 肺上皮細胞又は肺がん細胞からのオルガノイドの製造方法 | |
JP2022132249A (ja) | 多能性幹細胞の中腸内胚葉細胞への分化 | |
JP7357369B2 (ja) | 新規腎前駆細胞マーカーおよびそれを利用した腎前駆細胞の濃縮方法 | |
JP7274683B2 (ja) | 多能性幹細胞から樹状分岐した集合管を伴う腎臓構造を作製する方法 | |
JP7421049B2 (ja) | 糸球体ポドサイトの誘導方法、及び該誘導方法を用いた多能性幹細胞からのポドサイトの製造方法 | |
WO2017010448A1 (ja) | ネフロン形成能を有するネフロン前駆細胞の増幅培養方法 | |
KR101133553B1 (ko) | 줄기세포성 유지 또는 줄기세포 분화를 위한 줄기세포 배양용 조성물 | |
WO2023017848A1 (ja) | 腎間質前駆細胞の製造方法並びにエリスロポエチン産生細胞、およびレニン産生細胞の製造方法 | |
WO2020095423A1 (ja) | 多能性幹細胞から樹状分岐した集合管を伴う腎臓構造を作製する方法 | |
JP2013201943A (ja) | 成熟肝細胞の製造方法 | |
Zhou | Characterisation of Nascent Mesoderm Derived from Mouse Embryonic Stem Cells Grown in 3-D and 2-D Culture Systems | |
Waese | Design of a novel serum-free monolayer differentiation system for murine embryonic stem cell-derived chondrocytes for potential high-content imaging applications |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 14853879 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2015542663 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2014853879 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2014853879 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15026462 Country of ref document: US |