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EXPANSION CULTURE METHOD FOR NEPHRON PROGENITOR CELLS HAVING NEPHRON-FORMING CAPACITY

Foreign code F170009024
File No. (S2015-1763-N0)
Posted date Mar 29, 2017
Country WIPO
International application number 2016JP070396
International publication number WO 2017010448
Date of international filing Jul 11, 2016
Date of international publication Jan 19, 2017
Priority data
  • P2015-139271 (Jul 11, 2015) JP
  • P2016-076839 (Apr 6, 2016) JP
Title EXPANSION CULTURE METHOD FOR NEPHRON PROGENITOR CELLS HAVING NEPHRON-FORMING CAPACITY
Abstract The purpose of the present invention is to provide a method for the expansion culture of nephron progenitor cells while maintaining an undifferentiated state. Another purpose of the present invention is to provide an expansion culture method for cultured nephron progenitor cells to have nephron-forming ability. Yet another purpose of the present invention is to provide an expansion culture method for nephron progenitor cells that makes it possible to achieve an excellent expansion rate. The present invention provides a method for culturing and increasing nephron progenitor cells created from isolated mammalian nephron progenitor cells or iPS cells using a medium containing the following compounds: (i) Wnt agonist; (ii) Notch inhibitor; (iii) Bmp; (iv) leukemia inhibitory factor; (v) Rock inhibitor; and (vi) Fgf.
Outline of related art and contending technology BACKGROUND ART
Mammalian kidney, about 100 million nephrons are included in, it is, made of tubules and glomeruli. Adult kidney is an organ not displayed, the fetal period in the kidney, the metanephros tissue mesenchymal (mm) a transcription factor called Six2 positive progenitor cells present in the self-renewal of the nephron glomerular or tubules while repeating the major functional units of differentiating into the nephron. However, a progenitor cell, a mouse within several days post-natal development (kidney generated after from about 10) to, human 34 week gestation, self-stop replicating, to terminal differentiation. Therefore, new nephrons in the kidney of the adult form does not occur. This is, contributes to the adult kidney is not recovered and is considered to be. Then, but that cannot be recovered in the diseased kidney of the cause.
(Mm) is mesenchymal metanephros, nephron progenitor cells expressing the Six2 transcription factor comprises, these cells are fibroblasts derived from urinary tract induced by Wnt9 standard Wnt (tissue) in response to a signal to produce a nephron epithelium. Six2 Is, against Wnt-mediated differentiation signal and, thereby the nephron progenitor cells maintains the undifferentiated state. Therefore, the nephron progenitor cells, balance between self-replication and differentiation, important for kidney organogenesis.
Therefore, this can be differentiated into the nephron Six2 how positive the nephron progenitor cell populations and replicate, differentiate to understand whether the cell level, it is important that the kidney as a construction the base of regenerative medicine. In addition, a large amount of such precursor cells prepared kidney problems essential to regenerative medicine. Therefore, undifferentiated precursor cells cultured in a state of the nephron and amplification method has been tried up to now.
So far the nephron progenitor cells cultured in the undifferentiated state or amplification is performed a number of studies have been reported method. For example, amplification of the nephron progenitor cells of mice as reported in the attempt to culture is given as follows. Barak et al., 14-17 days embryonic mouse kidney from first Six2-GFP were isolated by FACS Six2-GFP positive cells, Bmp7 (50ng/ml), Fgf9 (8.6nm, 200ng/ml equivalent) and heparin (1μg/ml) were cultured under the culture conditions including (non-patent document 1). As a result, cells positive for 5 maintained between Six2-GFP and has succeeded. However, the proliferative capacity and the reduction of the culture and subsequent 2 capable of forming tubules are missing. On the other hand, Brown et al., mouse embryonic carcinoma Bmp7 cells positive for the first 17 isolated by FACS from the kidneys, and the cells are nephrogenic zone cell(NZC), nephron progenitor cell in a positive cells expressing Cited 1, Fgf-1, -2, each of the -9 and -20 in the presence of 24 (200ng/ml) is maintained at a time (non-patent document 2) reports. Further, Brown et al., (50ng/ml) and Bmp7 in the Wnt pathway activator (0.5μm) and the processing NZC BIO in differentiation (non-patent document 3) reports. These reports, maintenance of progenitor cells of the nephron Fgf(200ng/ml) /Bmp7(50ng/ml) is important on the other hand, by differentiation Wnt activator respectively. In addition, those reports, for the amplification amount of the nephron progenitor cells has not been described.
In addition, Yuri et al., re-aggregation of the nephron progenitor cells using the culture system ("re-aggregate"system), reported a method for amplifying (non-patent document 4). Where, mouse nephron Six2-GFP-positive progenitor cells, could be cultured up to 21, and 21 on the fourth Six2-GFP positive nephron progenitor cells greater than twice the number of successful amplification of the 20 is described. However, after 14, cell number is the plateau has been reached. In addition, and γ - secretase inhibiting Notch signaling pathway, is the nephron DAPT prevents differentiation of progenitor cells, the aggregates of the first new reconstructed from the aggregates in the nephron Six2-GFP positive effective maintenance of progenitor cells and amplification, the amplified up to 65 times the described Data. Yuri et al. in re-flocculation of the system, using the aggregates of the first, a single cell from the separated cells were prepared and, from there again is constructed with a new aggregates. However, after incubation of the cells, the aggregates of the epithelial marker of positive E-cadherin is shown, the glomerulus, tubules of the nephron of the three dimensions of the ability to reconstruct the structure not shown. In addition, this culture system is a 12 solution of trypsin EDTA 0.25% embryonic carcinoma detached in the kidney, ureter and fibroblast growth factor, progenitor cells and interstitial cells of the nephron are mixed and the resulting mixture of flocculating cells cultured. That is, the culture conditions Yuri et al., fibroblasts isolated from urinary tract nephron progenitor cells can be cultivated not only indicates. Therefore, this culture system is, embryonic stage is required and the sprouting of the urinary tract, and culture conditions for the amplification of the nephron progenitor cells act directly to whether or not is not clear.
As described above, the nephron progenitor cells, and that retain the ability to form nephrons while maintaining an undifferentiated state it is difficult to culture and amplifying, suitable culture method heretofore has not been established.
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION KUMAMOTO UNIVERSITY
  • Inventor
  • NISHINAKAMURA, Ryuichi
  • TANIGAWA, Shunsuke
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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