WO2017010448A1 - Procédé de culture d'expansion de cellules progénitrices de néphron présentant la capacité de former des néphrons - Google Patents

Procédé de culture d'expansion de cellules progénitrices de néphron présentant la capacité de former des néphrons Download PDF

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WO2017010448A1
WO2017010448A1 PCT/JP2016/070396 JP2016070396W WO2017010448A1 WO 2017010448 A1 WO2017010448 A1 WO 2017010448A1 JP 2016070396 W JP2016070396 W JP 2016070396W WO 2017010448 A1 WO2017010448 A1 WO 2017010448A1
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cells
progenitor cells
nephron
medium
concentration
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隆一 西中村
俊祐 谷川
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国立大学法人熊本大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention relates to an amplification culture method for nephron progenitor cells. More specifically, the present invention relates to a culture method capable of amplifying nephron progenitor cells while maintaining an undifferentiated state without losing differentiation ability.
  • the mammalian kidney contains about 1 million nephrons, which consist of glomeruli and tubules.
  • the adult kidney is an organ that does not regenerate, but in the fetal kidney, there is a transcription factor Six2-positive nephron progenitor cell in a tissue called the metanephric mesenchyme (MM), and it repeats self-replication from glomeruli and tubules. It differentiates into nephron, which is a major functional unit.
  • progenitor cells stop self-replicating and terminally differentiate within a few days after birth in mice (approximately 10 days after kidney development) and at 34 weeks of gestation in humans. Therefore, no new nephron formation occurs in the adult kidney. This is thought to be one reason why the adult kidney does not regenerate. And it causes the pathological kidney to be unrecoverable.
  • the metanephric mesenchyme contains nephron progenitor cells that express the transcription factor Six2, which respond to the standard Wnt signal induced by ureteric bud-derived Wnt9 in the nephron epithelium (tissue). Produce. Six2 counters Wnt-mediated differentiation signals, thereby maintaining nephron progenitor cells in an undifferentiated state.
  • Six2 counters Wnt-mediated differentiation signals, thereby maintaining nephron progenitor cells in an undifferentiated state.
  • the balance between self-renewal and differentiation of nephron progenitor cells is important for renal organogenesis.
  • Brown et al. Isolated Bmp7-positive cells from the kidney of embryonic day 17 of mice by FACS. The cells were nephrogenic zone cells (NZC), and Cited1 positive cells expressed in nephron progenitor cells were Fgf- It has been reported that it is maintained for 24 hours in the presence of 1, -2, -9 and -20 (200 ng / ml) (Non-patent Document 2). Furthermore, Brown et al.
  • NZC is differentiated by treatment with Bmp7 (50 ng / ml) and BIO (0.5 ⁇ M), an activator of the Wnt pathway.
  • BIO an activator of the Wnt pathway
  • Non-Patent Document 4 Yuri et al. Also reported a method for culturing and amplifying nephron progenitor cells using a reaggregation system. It describes that Six2-GFP positive mouse nephron progenitor cells could be cultured until day 21 and that the number of Six2-GFP positive nephron progenitor cells was amplified more than 20 times on day 21. Has been. However, after 14 days, the cell count has reached a plateau.
  • DAPT which is a ⁇ -secretase that inhibits Notch signaling pathway and prevents nephron progenitor cell differentiation, maintains Six2-GFP positive nephron progenitor cells in new aggregates reconstructed from the initial aggregates. It is effective for amplification and is described as being able to amplify up to 65 times.
  • Cells separated into single cells are prepared using the initial aggregate, and a new aggregate is constructed again therefrom.
  • cells after culture have been shown to be positive for the epithelial marker E-cadherin, but the ability to reconstruct the three-dimensional nephron structure of glomeruli and tubules has not been shown.
  • the embryonic day 12 kidney was dissociated with a 0.25% trypsin EDTA solution, and cells in which ureteric buds, nephron progenitor cells and interstitial cells were mixed were cultured. Yes. That is, it is shown that the culture conditions of Yuri et al. Cannot be cultured only with nephron progenitor cells isolated from ureteric buds. Therefore, this culture system requires embryonic ureteric buds, and it is not clear whether the culture conditions directly affect nephron progenitor cell amplification.
  • the present inventors have obtained a high rate of Six2-positive nephron progenitor cells having the ability to form a three-dimensional kidney tissue of glomeruli and tubules (for example, Nephron progenitor cells are maintained for a long period of time (for example, at least 7 days or more, preferably 10 days or more) while maintaining 60% or more of the whole, preferably 70% or more, more preferably 80% or more, more preferably 90% or more. (More preferably 15 days or more, and even more preferably 19 days or more) a culture method for amplification was established, and the present invention was completed.
  • a culture method for amplification was established, and the present invention was completed.
  • the present inventors added the concentration of Wnt activator in the medium and the concentration of Bmp7 in the medium in addition to the addition of Fgf9, Bmp7, CHIR99021 to the medium.
  • the combination was found to be important for the differentiation and proliferation of nephron progenitor cells.
  • the present inventors have found that Wnt activator and Bmp7 alone are not sufficient for maintaining highly efficient Six2-positive cells, and that Lif is essential.
  • the present invention includes the following.
  • the following compounds (I) a Wnt agonist, (Ii) Fgf, (Iii) leukocyte inhibitory factor (LIF), (Iv) Bmp family compounds, A method for amplifying and culturing nephron progenitor cells derived from an isolated mammal using a medium containing (v) a Rock inhibitor and (vi) a Notch inhibitor.
  • the medium further contains (vii) Tgf- ⁇ .
  • the medium contains a Bmp family compound at a concentration of 1.0 ng / ml to 20 ng / ml (preferably 2.5 ng / ml to 15 ng / ml, more preferably 5 ng / ml to 15 ng / ml).
  • a Bmp family compound at a concentration of 1.0 ng / ml to 20 ng / ml (preferably 2.5 ng / ml to 15 ng / ml, more preferably 5 ng / ml to 15 ng / ml).
  • the medium is a Wnt agonist at a concentration of 0.1 ⁇ M to 5 ⁇ M (preferably 0.5 ⁇ M to 2.5 ⁇ M, more preferably 0.75 ⁇ M to 1.5 ⁇ M), 1.0 ng / ml to 20 ng / ml (Preferably 2.5 ng / ml to 15 ng / ml, more preferably 5 ng / ml to 15 ng / ml) Bmp family compound at a concentration of 10 to 500 ng / ml (preferably 30 to 300 ng / ml, more preferably Fgf at a concentration of 50 to 200 ng / ml) and LIF at a concentration of 1 ng / ml to 30 ng / ml (preferably 1 ng / ml to 20 ng / ml, more preferably 1 ng / ml to 10 ng / ml)
  • the method according to any one of [1] to [3] above, comprising:
  • the Wnt agonist is a GSK-3 inhibitor (preferably CHIR99021, BIO, or SB415286, more preferably CHIR99021).
  • the Fgf is selected from the group consisting of Fgf1, Fgf2, Fgf9, and Fgf20.
  • the Wnt agonist is CHIR99021, the Fgf is Fgf9 or Fgf2, the Bmp is Bmp7, the Rock inhibitor is Y27632, and the Notch inhibitor is DAPT.
  • the nephron progenitor cell is an ES cell or iPS cell-derived nephron progenitor cell (preferably a mouse ES cell or mouse iPS cell-derived mouse nephron progenitor cell, or a human ES cell or human iPS cell-derived human nephron progenitor cell.
  • the method according to any one of [1] to [10] above.
  • [12] The above [1] to [11], wherein the nephron progenitor cells are cultured for 5 days or more (preferably 7 days or more, more preferably 10 days or more, still more preferably 15 days or more). The method according to any one of the above.
  • the nephron progenitor cells are 4 times or more (preferably 10 times or more, more preferably 30 times or more, still more preferably 50 times or more, most preferably 100 times or more) compared to the number of cells at the start of culture.
  • the culture is performed on an iMatrix or fibronectin-coated plate.
  • the culturing is performed in a U-bottom low cell adsorption plate.
  • [16] A nephron progenitor cell having nephron-forming ability, cultured using the method according to any one of [1] to [15] above.
  • [17] A method for producing a three-dimensional kidney structure having glomeruli and tubules using nephron progenitor cells cultured using the method according to any one of [1] to [15] above.
  • Wnt agonist at a concentration of 0.1 ⁇ M to 5 ⁇ M (preferably 0.5 ⁇ M to 2.5 ⁇ M, more preferably 0.75 ⁇ M to 1.5 ⁇ M), 1.0 ng / ml to 20 ng / ml (preferably Bmp family compounds at a concentration of 2.5 ng / ml to 15 ng / ml, more preferably 5 ng / ml to 15 ng / ml), 10 to 500 ng / ml (preferably 30 to 300 ng / ml, more preferably 50 to 200 g / ml) and LIF at a concentration of 1 ng / ml to 30 ng / ml (preferably 1 ng / ml to 20 ng / ml, more preferably 1 ng / ml to 10 ng / ml), [18] The medium according to any one of [20] to [20].
  • the medium comprises a Rock inhibitor at a concentration of 1.0 ⁇ M to 50 ⁇ M (preferably 2.0 ⁇ M to 30 ⁇ M, more preferably 5 ⁇ M to 20 ⁇ M), and 1.0 ⁇ M to 20 ⁇ M (preferably 2.
  • the Wnt agonist is CHIR99021
  • the Fgf is Fgf9 or Fgf2
  • the Bmp is Bmp7
  • the Rock inhibitor is Y27632
  • the Notch inhibitor is DAPT.
  • nephron progenitor cells can be cultured and expanded while being maintained in an undifferentiated state, and the nephron progenitor cells thus increased have the ability to differentiate into nephrons. That is, according to the present invention, amplification culture of nephron progenitor cells has become possible.
  • FIG. 5 is the result of qPCR analysis of MM nephron progenitor cell markers cultured on plates coated with iMatrix or fibronectin (Fn) for 7 days. MM freshly isolated at E11.5 was used as a positive control (E11.5MM).
  • FT Control; LY: LIF + Y27632; LYD: LY + DAPT. It is the result of having culture
  • FIG. 6 is the result of reconstitution of 3D nephron structure from cells grown using cells prepared from E15.5 or P0 kidneys.
  • FIG. 6 shows the results of confirming the influence of FGF2 and FGF9 on the proliferation of cells from kidneys of E11.5 Six2-GFP mice.
  • GFP positive cells from E11.5 were cultured for 7 days in FGF2 (50 ng / ml) or FGF9 (50 ng / ml) and then analyzed by FACS.
  • the results of FACS analysis at the start of culture are shown in the upper left panel, and the results of FACS analysis after 7 days of culture are shown in the upper right panel.
  • the lower left panel shows the total number of cells and the number of Six2-GFP positive cells at the start of culture (E11.5) and on the seventh day of culture.
  • the lower right panel shows the expression of nephron progenitor cell markers (Six2, Pax2, Sall1, and Wt1) in each condition.
  • FIG. 6 is a result of reconstitution of three-dimensional nephron structure from cells grown using cells prepared from E11.5 kidneys.
  • Left row hematoxylin and eosin (HE) staining; middle row: immunostaining for nephrin (red) and WT1 (green), markers for glomeruli; right row: markers for distal end and proximal tubule, respectively Immunostaining of Cadherin 1 (red) and Cadherin 6 (green).
  • Scale bar 20 ⁇ m.
  • White arrows indicate glomeruli, and black arrows indicate tubules. The examination of the optimal concentration of CHIR and Bmp7 in the proliferation of nephron progenitor cells is shown.
  • FIG. 2 is a schematic diagram of continuous passage culture of Six2-GFP positive cells from E11.5.
  • Sorted progenitor cells (green) were cultured for 2 days to aggregate, and further cultured on plates for 6 days (day 8). Cells were then diluted 1: 3 and cultured until day 19. The total number of cells on day 19 of culture and the number of GFP positive cells in optimized conditions (including all factors) or in the absence of the indicated factors are shown. The percentage of GFP positive cells in culture is shown.
  • the result of the quantitative PCR analysis of a nephron progenitor cell marker (Six2, Pax2, Sall1, Wt1, and Osr1) is shown. It is the result of reconstruction of the three-dimensional nephron structure from cells cultured for 19 days.
  • HE hematoxylin and eosin staining
  • middle row immunostaining for nephrin (red) and WT1 (green), markers for glomeruli
  • right row markers for distal end and proximal tubule, respectively Immunostaining of Cadherin 1 (red) and Cadherin 6 (green).
  • Scale bar 20 ⁇ m.
  • White arrows indicate glomeruli, and black arrows indicate tubules. It is the result of calculating the number of amplified cells of nephron progenitor cells in vitro compared with the state in vivo.
  • Right panel calculated number of GFP positive cells cultured in vitro for 8 and 19 days from one E11.5 kidney progenitor cell. Shown are the results of quantitative PCR analysis (right panel) of the total number of cells and the number of OSR1-GFP positive cells (left panel) and nephron progenitor cell markers (Six2, Pax2, Sall1, Wt1, and Osr1) on day 7 of culture. Yes. It is the result of reconstitution of nephron structure from nephron progenitor cells cultured for 7 days from OSR1-GFP ES cells.
  • the left figure shows the results of measuring the number of nephron progenitor cells on day 0 and 8 of culture, and the right figure shows the results of measuring the expression level of nephron progenitor cell markers. It is a result of reconstruction of a three-dimensional nephron structure from cells grown using nephron progenitor cells derived from human iPS cells.
  • Left column HE staining; middle column: immunostaining of nephrin (red) and WT1 (green) markers for glomeruli; right column: Cadherin1 (marker for distal end and proximal tubule, respectively) Red) and Cadherin 6 (green) immunostaining.
  • Scale bar 20 ⁇ m. Two white arrows on the right side indicate glomeruli, and two black arrows on the left side indicate tubules.
  • One embodiment of the present invention provides for nephron progenitor cells comprising (i) a Wnt agonist, (ii) Fgf, (iii) a leukocyte inhibitory factor (LIF), (iv) a Bmp family compound, (v) a Rock inhibitor, and ( vi) It is a method of increasing by culturing in a medium containing a Notch inhibitor. Another aspect of the present invention is a method of increasing nephron progenitor cells by further culturing in a medium containing (vii) Tgfa. Another aspect of the present invention is a medium that can amplify and culture nephron progenitor cells in an undifferentiated state.
  • nephron progenitor cell having nephron-forming ability cultured by the above method.
  • nephron-forming ability means that a three-dimensional nephron structure having differentiated and developed glomeruli and tubules can be formed.
  • Another embodiment of the present invention is a method for producing a three-dimensional kidney structure (nephron) having glomeruli and tubules using nephron progenitor cells amplified and cultured by the method of the present invention.
  • the medium used in the culture method of the present invention can be a commonly used medium as a basal medium, and is not particularly limited as long as the object of the present invention can be achieved.
  • a medium used for animal cell culture is a basal medium.
  • the basal medium for example, BME medium, BGjB medium, CMRL61066 medium, Glasgow MEM medium, improved MEM medium, IMDM medium, Medium 199 medium, Eagles MEM medium, ⁇ MEM medium, DMEM medium, ham medium, RPMI401640 medium, Fischer Examples thereof include 's medium, Dulbecco medium, improved Dulbecco medium, and mixed media thereof.
  • a DMEM / F12 medium obtained by adding F12 to DMEM can be used, but is not limited thereto.
  • 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.
  • the method for amplifying and culturing nephron progenitor cells of the present invention can be carried out according to the present invention using a medium obtained by adding each component or factor to the above medium, as described in detail later.
  • the components and factors arbitrarily 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.
  • Wnt agonist 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 and valproic acid, benzazepineone family Kenpaulone; 9-bromo-7,12-dihydroindolo [3,2-d] [1] benzacepine-6 (5H)- ON) and Alster Paulon (Alsterpaullone; 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-
  • 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.
  • 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.
  • These Wnt agonists are commercially available.
  • the concentration in the medium can be, for example, 0.1 to 5.0 ⁇ M, preferably 0.5 to 2.5 ⁇ M, more preferably 0.75 to 1.5 ⁇ M. .
  • the Fgf that can be used in the present invention can be selected from the group consisting of the Fgf family, preferably selected from Fgf2, Fgf9, and Fgf20, and more preferably Fgf9.
  • the concentration of Fgf in the medium is, for example, 10 to 500 ng / ml, preferably 30 to 300 ng / ml, more preferably 50 to 200 ng / ml.
  • these Fgf are also marketed and can be used for this invention without a restriction
  • LIF Leukocyte inhibitor
  • concentration of LIF used in the present invention in the medium is, for example, 1.0 ng / ml to 30 ng / ml, preferably 1.0 ng / ml to 20 ng / ml, more preferably 1.0 ng / ml to 10 ng / ml.
  • concentration of ml can be increased.
  • High concentrations of LIF eg, 50 ng / ml or higher
  • PLC phospholipase C
  • Jnks c-Jun N-terminal kinase
  • Such activation is mild at low concentrations, and low concentrations of LIF can maintain nuclear localization of Six2 and Yap, which are important for progenitor cell amplification.
  • combined treatment with a Rock inhibitor (Y27632) attenuates LIF-induced JNK activation, thereby allowing nephron progenitor cell amplification.
  • Y27632 Rock inhibitor
  • Bmp The Bmp family compound used in the present invention (hereinafter sometimes simply referred to as “Bmp”) is selected from the group consisting of Bmp family such as Bmp1, Bmp2, Bmp4, Bmp6, Bmp7, Bmp8a, Bmp8b and Bmp10, GDF11. , Preferably selected from Bmp2, Bmp4 or Bmp7, more preferably Bmp7. These Bmps are commercially available. The concentration of Bmp in the medium can be appropriately selected depending on the type of Bmp.
  • Bmp7 when used, for example, 1.0 ng / ml to 20 ng / ml, preferably 2.5 ng / ml to 15 ng / ml, more preferably 5 ng / ml to 15 ng / ml. These concentrations are very different from the concentrations (50 ng / ml) used in the cited prior art (Non-Patent Documents 1 to 4).
  • the Bmp to be used can be any source of Bmp, but is preferably human Bmp. When other Bmp compounds are used, the concentration at which the same effect as that obtained when Bmp7 is used can be appropriately selected.
  • Rock inhibitor Rock is a serine / threonine protein kinase that has been identified as a target protein for the low molecular weight GTP binding protein Rho.
  • Rock inhibitors that are inhibitors thereof are commercially available, and those can be used without limitation in the present invention.
  • Examples of the Rock inhibitor used in the present invention include, but are not limited to, Y27632 and HA1077, and Y27362 is preferable.
  • the concentration of the Rock inhibitor used in the present invention can be, for example, 1 to 50 ⁇ M, preferably 2 to 30 ⁇ M, more preferably 5 to 20 ⁇ M.
  • the Notch inhibitor used in the present invention is a compound that inhibits Notch signal, and ⁇ -selectase is also included in the Notch signal inhibitor of the present invention as long as it exhibits the same activity.
  • a preferred Notch inhibitor includes DAPT, which is commercially available.
  • the concentration of the Notch inhibitor in the medium can be appropriately selected according to the type of Notch inhibitor to be used. For example, when DAPT is used, for example, 1.0 to 20 ⁇ M, preferably 1.0 to 10 ⁇ M, More preferably, the concentration is 1.0 to 5 ⁇ M, more preferably 2.5 to 5 ⁇ M.
  • Tgf ⁇ can be further added to the medium.
  • Tgf ⁇ is a factor generally used in the culture of undifferentiated cells such as ES cells and iPS cells, and is a factor usually added to the medium in the culture of nephron progenitor cells.
  • the concentration of Tgf ⁇ in the medium can be, for example, 1 to 500 ng / ml, preferably 5 to 300 ng / ml, more preferably 10 to 200 ng / ml.
  • These Tgfas are also commercially available and can be used in the present invention without any particular limitation.
  • cells can be cultured on a matrix-coated plate, and it is expected to obtain better culture results.
  • Coating matrices include, but are not limited to, iMatrix, fibronectin, laminin, and collagen, and plates coated with these matrices are commercially available.
  • the cells can be cultured in a spheroid-like U-bottom plate (for example, a low-cell-binding-plate having a plurality of wells), and a better amplification culture can be achieved.
  • One aspect of the method of the present invention is to culture the cells while maintaining the spheroid form.
  • Nephron progenitor cells used in the culturing method of the present invention may be any of nephron progenitor cells collected and collected from living bodies and nephron progenitor cells induced to differentiate from pluripotent stem cells (for example, ES cells and iPS cells). Can be used.
  • the collection and collection of mammalian nephron progenitor cells other than humans from living bodies can be performed by removing the fetal kidney and isolating the metanephric mesenchyme.
  • nephron progenitor cells of the mouse on the 12th day to the 1st day after birth can be isolated and collected by FACS.
  • Nephron progenitor cells from ES cells or iPS cells can be prepared, for example, according to the report of the present inventors (Non-patent Document 6; Patent Document 1).
  • the progenitor cells can continue to proliferate and are at least 5 days, preferably 7 days, more preferably 10 days or more, even more preferably 12 days or more, and even more preferably Culturing is possible for 15 days or more, most preferably 19 days or more.
  • the number of cells is at least 10 times or more, usually 30 times or more, preferably 50, compared to the time of initiation of culture.
  • Amplification of at least twice, more preferably at least 70 times, even more preferably at least 100 times, and even more preferably at least 1000 times is possible.
  • the number of cells can be amplified by at least 4 times, preferably 10 times or more, compared to the time at the start of culture.
  • Nephron progenitor cells cultured by the amplification culture method of the present invention maintain an undifferentiated state and have the ability to differentiate into nephrons.
  • nephron progenitor cells are purified and then nephron efficiently using the amplification culture method of the present invention.
  • Progenitor cells can be prepared.
  • nephron progenitor cells are isolated from tissue derived from human iPS cells, and after purifying the cells, nephron progenitor cells having nephron-forming ability are amplified and cultured using the amplification culture method of the present invention. be able to.
  • the cell mass (cell aggregate) is separated and divided into a plurality of small cell masses during the culture, for example, about 7 days, and then the culture is continued.
  • progenitor cells can be efficiently amplified.
  • nephron progenitor cells cultured by the amplification culture method of the present invention can be stored frozen.
  • it can be cryopreserved using an existing cell cryopreservation solution, and the nephron progenitor cells thus stored can be used to reconstitute a three-dimensional nephron structure (eg, glomeruli and urine). (Formation of a thin tube) is possible.
  • the isolated MM is trypsinized (0.25% trypsin / EDTA), washed with buffer, FACS buffer (1 ⁇ HBSS, 1% BSA, 0.035% NaHCO 3 , and 1 ⁇ g / ml iodinated) Suspended in propidium).
  • Isolation of E15.5 Six2-GFP positive cells was performed by incubating kidneys with 0.25% trypsin / EDTA for 10 minutes at 37 ° C. and separating the cells by pipetting in cold DMEM containing 10% FCS. .
  • Neonatal kidneys are treated with a mixture of collagenase XI (Sigma-Aldrich), dispase (Life Technologies), and DNase (Roche) for 10 minutes at 37 ° C. and then with 0.25% trypsin-EDTA. The cells were treated at 37 ° C. for 5 minutes and separated into single cells.
  • Serum DMEM / F12 (Life Technologies) was used (abbreviated as “FT”).
  • FT Serum DMEM / F12
  • Optimum conditions (abbreviated as “CDBLY”) are further: 1 ⁇ M CHIR99021 (Wako), 2.5 ⁇ M DAPT (Merck Millipore), 5 ng / ml BMP7 (R & D Systems), 5 ng / ml LIF (Millpore) , And 10 ⁇ M Y27632 (Wako).
  • OSR1-GFP mouse ES cells were used for induction of nephron progenitor cells.
  • ES cells were passaged on gelatin coated plates for 2 days to remove feeders. Cells were then harvested, aggregated and differentiated with Accutase (Merck Millipore) according to the manufacturer's 5-step protocol. Induced spheroids were collected at day 8.5 and dissociated using 0.25% trypsin EDTA. Cells were blocked with normal mouse serum and stained with anti-integrin ⁇ 8 (R & D Systems) and anti-PDGFR ⁇ (Cell signaling) antibodies in FACS buffer.
  • OSR1-GFP + / integrin ⁇ 8 + / Pdgfr ⁇ fractions were sorted with FACSAria SORP (BD) and cultured in a medium supplemented with CDBLY.
  • nephron structure prepared from cultured mouse progenitor cells Cultured cells were seeded on low-cell-binding plates and allowed to form aggregates over 48 hours. Then, on the Nucleopore membrane (Millipore) suspended in DMEM medium containing 10% FCS, the cells were co-cultured with the spinal cord of the embryo for 7 days. The medium was changed every 3 days. The cultured graft was fixed with 10% formalin and embedded in paraffin. Sliced into 6 micron sections, deparaffinized, and then antigen retrieval with citric acid (10 mM, pH 6.0) at 121 ° C. for 5 minutes. Sections were blocked with 1% bovine serum albumin (BSA), incubated with primary antibody at 4 ° C.
  • BSA bovine serum albumin
  • the primary antibodies used are as follows: anti-cadherin 1 (BD), anti-cadherin 6 (provided by Dr. Gregory Dressler, Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA), anti-nephrin (Progen), And anti-WT1 (Santa Cruz). Secondary antibodies used are as follows: anti-rabbit Alexa 488, anti-guinea pig Alexa 468, and anti-mouse Alexa 594 (Life Technologies).
  • Primer sequences for qPCR for each marker gene ( ⁇ -Actin, E-Cadherin, Lef1, Osr1, Pax2, Sall1, Six2, and Wnt1) were purchased from Takara Bio. Incubation conditions were 45 cycles (95 ° C. for 15 seconds, 60 ° C. for 45 seconds). All samples were normalized with actin expression.
  • Example Example 1 Total MM was isolated from the kidneys of Six2-GFP mice at E11.5 in order to accurately determine the proportion of mouse nephron progenitor cells during partial amplification culture of nephron progenitor cells from the metanephric mesenchyme.
  • LIF and Y27632 collectively abbreviated as “LY”
  • the cells were cultured as a cell mass without being dispersed into individual cells in the presence of LIF and Y27632 (collectively abbreviated as “LY”). 1).
  • LY LIF and Y27632
  • CHIR99021 the Wnt agonist CHIR99021 (CHIR) was tested because Wnt signaling plays an important role in the maintenance and differentiation of nephron progenitor cells.
  • CHIR increased GFP positive cells on day 7 of culture and the combination of LIF and CHIR was A large amount of amplification of GFP positive cells was shown. Therefore, a combination of LIF, Y27632, CHIR, and DAPT (abbreviated as “CDLY”) was tested on an iMatrix-coated plate. As a result, 26.3% became positive after 7 days of culture.
  • BMP7 which is an important regulator of nephron progenitor cells.
  • nephron progenitor cell markers including Six, Pax2, Sall1, Wt1, and Osr1
  • CDBLY abbreviated as “CDBLY”. It was almost the same. Therefore, although the culture was started from the whole MM including the mixed cell population, this condition was found to be suitable for the amplification of nephron progenitor cells.
  • Example 2 In Vitro Amplification of Nephron Progenitor Cells Purified from Mouse Embryos and Neonatal Kidneys
  • the above culture conditions (CDBLY) were applied to purified Six2-GFP positive progenitor cells. Due to cell number limitations, the results were initially sorted using E15.5 and newborn (P0) kidney instead of E11.5, resulting in 21.1% and 8% of total GFP positive cells, respectively. It was 1%. The sorted cells were then cultured and aggregated for 2 days to form spheroids, seeded on iMatrix coated plates and cultured for 5 days or longer under the conditions described above. As shown in FIG.
  • the glomeruli were round and positive for podocyte-specific markers, Wt1 and nephrin. Induced tubules were localized into cadherin 6 positive proximal and cadherin 1 positive distal domains.
  • this culture condition allowed the growth of purified nephron progenitor cells while maintaining the ability to form the three-dimensional nephron structure shown by well-developed glomeruli and tubules.
  • progenitor cells from newborns were maintained for 7 days. This is because mouse nephron progenitor cells stop proliferating within 2-3 days after birth and undergo terminal differentiation. Therefore, this result shows that the culture conditions of the present invention maintain the proliferation of progenitor cells in vivo exceeding their physiological time limit.
  • Example 3 Effect of LIF, FGF2 / 9, BMP7, and Wnt agonist on the proliferation of mouse nephron progenitor cells
  • the above culture conditions allow the proliferation of purified nephron progenitor cells from E15.5 or P0, but cell number The increase was not dramatic (approximately double, see FIG. 4).
  • the culture conditions were optimized using all MM from E11.5, and it was confirmed that similar results were obtained with E15.5 and P0.
  • Six2-GFP positive progenitor cells were purified with E11.5.
  • the sorted cells were aggregated and cultured on an iMatrix-coated plate for 7 days.
  • the medium used contained Fgf2 in all experiments. This is based on the results of rat MM culture recently reported by the inventors. Since Fgf9 has been reported to be required for the maintenance of mouse nephron progenitor cells in vivo, the effects of Fgf2 and Fgf9 in culture were compared. Both Fgf2 and Fgdf9 maintained Six2-GFP positive cells at a very high rate (> 95%), but a larger increase in cell number was observed with Fgf9 based conditions (30-fold vs. 60-fold). (FIG. 6).
  • nephron progenitor cell markers (Six2, Pax2, Sall1, and Wt1) was equivalent to that of Six2-GFP positive cells newly isolated in E11.5 (FIG. 6). . As shown in FIG. 7, glomeruli and tubules were formed under any conditions when combined with the spinal cord. Therefore, Fgf9 was used in the following experiment.
  • Example 4 Reconstruction of three-dimensional nephron structure using mouse embryonic nephron progenitor cells passaged for 19 days
  • the cells were separated into small clumps (aggregates) and divided into 3 plates the cells continued to grow and could be passaged every 3-4 days until day 19 (see FIG. 10). .
  • the cell number increased and 97% remained positive for Six2-GFP (FIG. 11). Under the optimum conditions (addition of all factors), the percentage of GFP-positive cells on day 19 was 97%.
  • mouse fetal nephron progenitor cells were amplified about 1500 times in 19 days of culture while maintaining an undifferentiated state, and the amplified progenitor cells were also threaded after culture. It was found that a three-dimensional structure of a sphere and a tubule can be formed.
  • Example 5 Amplification of nephron progenitor cells prepared from mouse ES cells and nephron forming ability
  • the inventors have recently reported the preparation of nephron progenitor cells from mouse ES cells (Non-patent Document 6; Patent Document 1). . Therefore, it was examined whether the above-described amplification culture method can be applied to ES cell-derived nephron progenitor cells. Since Six2-GFP ES cells capable of differentiating into the kidney lineage were not available, nephron progenitor cells and subsequently Osr1-GFP ES cells that were shown to differentiate into three-dimensional nephrons were used.
  • Osr1 is expressed not only in nephron progenitor cells but also in the stroma at this stage, but its expression is restricted to nephron progenitor cells at later stages of development. Therefore, the Osr1-GFP + / integrin ⁇ 8 + / Pdgfr ⁇ population was selected as the nephron progenitor cell fraction and cultured for 7 days. Cells grew 15-fold and 90.3 percent of them remained positive for Osr1-GFP, but the proportion of integrin ⁇ 8 + / Pdgfr ⁇ - fraction was reduced to 65.2 percent.
  • nephron progenitor cell markers (Six2, Pax2, Sall1, Wt1 and Osr1) in Osr1-GFP positive cells on day 7 was comparable to that of Six2 positive cells in vivo at E11.5. These cultured cells formed a three-dimensional nephron structure when stimulated by the spinal cord (FIG. 17). Therefore, nephron progenitor cells prepared from mouse ES cells can be amplified while maintaining nephron-forming ability, which indicates that the culture conditions of the present invention are robust.
  • nephron progenitor cells derived from mouse ES cells were amplified 15 times in 7 days of culture by the method of the present invention, and the amplified cells could form a three-dimensional structure of glomeruli and tubules. .
  • Example 6 Amplification of nephron progenitor cells prepared from human iPS cells and nephron formation Nephron progenitor cells are induced from human iPS cells according to the method of Taiguchi et al. At that time, human iPS cells whose nephron progenitor cell marker glows with GFP are prepared, and nephron progenitor cells are isolated by FACS. Thereafter, nephron cells are cultured at the concentration of each compound described in the present invention in the same manner as in the above Examples.
  • Non-patent Document 6 Using the cultured nephron progenitor cells, a three-dimensional nephron structure is formed in the same manner as in the above example, according to the known report of the inventor (Non-patent Document 6; Patent Document 1). Specifically, it was performed as follows. The outline is shown in FIG. Human nephron progenitor cells were prepared from iPS cells (201B7) according to the method of Taiguchi et al. The precursor spheroids on day 14 of culture were treated with a dissociation solution (CTK solution, Reprocell) to dissociate into small lumps.
  • CTK solution dissociation solution
  • FIG. 19 shows the results of measuring the number of nephron precursor cells and the gene expression level of the nephron precursor cell marker on day 8 of culture. Cells were amplified 4 times in 8 days.
  • a differentiation assay into nephron was performed.
  • the iPS cell-derived nephron precursor cells on day 8 of culture were dissociated into small clumps using a dissociation solution (CTK solution) and seeded in 96-well U-bottom low-cell-binding plates (200,000 cells per well) ) Incubated for 24-48 hours to form aggregates and then co-cultured with embryonic spinal cord to form nephrons. The result is shown in FIG. Glomeruli and tubules were confirmed.
  • Example 7 Nephron formation from human iPS cell-derived nephron progenitor cells
  • amplification of human iPS cell-derived nephron progenitor cells and nephron formation of the amplified cells were performed.
  • the precursor spheroids on the 14th day of culture were divided into 4 with a tungsten needle using 96-well U-bottom low-cell-binding plates instead of iMartin-coated plates, and cultured in a spheroid form. .
  • nephrons were formed, and many glomeruli and tubules were confirmed (data not shown).
  • Example 8 Amplification of nephron progenitor cells derived from purified human iPS cells
  • a tissue containing spheroid-like nephron progenitor cells derived from cultured human iPS cells 201B7 according to the method of Taiguchi et al. was immersed in 1x Accumax cell detachment solution (Millipore). , Treated at 37 ° C. for 8 minutes and dissociated (20 spheroids / 500 ⁇ L of Accumax cell detachment solution). Meanwhile, the solution was stirred every 2 minutes. Immediately after the reaction, centrifugation was performed (1000 rpm, 2 minutes).
  • the method of the present invention is useful as an amplification culture method for nephron progenitor cells. Further, nephron progenitor cells amplified and cultured by the method of the present invention are useful as research materials for neurogenesis and regeneration, and as raw materials for regenerative medicine.

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Abstract

L'objet de la présente invention est de fournir un procédé de culture d'expansion de cellules progénitrices de néphron tout en maintenant un état indifférencié. Un autre objet de la présente invention est de fournir un procédé de culture d'expansion de cellules progénitrices de néphron cultivées pour avoir la capacité de former des néphrons. Encore un autre objet de la présente invention est de fournir un procédé de culture d'expansion de cellules progénitrices de néphron qui permet d'obtenir un excellent taux d'expansion. La présente invention concerne un procédé permettant de cultiver et d'augmenter des cellules progénitrices de néphron créées à partir de cellules progénitrices de néphron de mammifères isolées ou de cellules souches pluripotentes induites (CSPI) à l'aide d'un milieu contenant les composés suivants : (i) un agoniste de Wnt ; (ii) un inhibiteur de Notch ; (iii) une Bmp ; (iv) le facteur inhibiteur de la leucémie ; (v) un inhibiteur de Rock ; et (vi) un Fgf.
PCT/JP2016/070396 2015-07-11 2016-07-11 Procédé de culture d'expansion de cellules progénitrices de néphron présentant la capacité de former des néphrons WO2017010448A1 (fr)

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WO2022149615A1 (fr) * 2021-01-08 2022-07-14 国立大学法人京都大学 Milieu de culture pour la culture d'expansion de cellule progénitrice de néphron, procédé de réalisation d'une culture d'expansion de cellule progénitrice de néphron et procédé de production d'organoïde de rein
WO2022149616A1 (fr) * 2021-01-08 2022-07-14 国立大学法人京都大学 Milieu de culture et de multiplication de cellules progénitrices de néphron, procédé de culture et de multiplication de cellules progénitrices néphroniques, et procédé de production d'organoïdes rénaux

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