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Inducer for urinary tract epithelial cells and inducing method for urinary tract epithelial cells

A technology of urothelial cells and cells, applied in the direction of urinary tract/kidney cells, cell culture active agents, botany equipment and methods, etc., can solve the problems of transformation into urothelial cells that have not been reported, and achieve the effect of avoiding canceration

Pending Publication Date: 2022-06-24
세루아쿠시아가부시키가이샤
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventors of the present application have previously determined and reported direct recombination of genes from somatic cells (for example, fibroblasts) into osteoblasts (patent document 1, non-patent document 5), brown adipocytes (patent document 2, non-patent document 6) , Schwann cell (patent document 3, non-patent document 7) and myoblast (patent document 4, non-patent document 8), however, there is no report of transformation from somatic cells to urothelium Cell example

Method used

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  • Inducer for urinary tract epithelial cells and inducing method for urinary tract epithelial cells
  • Inducer for urinary tract epithelial cells and inducing method for urinary tract epithelial cells
  • Inducer for urinary tract epithelial cells and inducing method for urinary tract epithelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0296] Adult dermal fibroblasts (aHDFs) are induced to express urothelial-like Phenotype.

[0297] Initially, we focused on three transcription factors. FOXA1(F) and IRF1(I) are important in the development of urothelial cells. On the other hand, TP63(T) is a transcription factor expressed in epithelial stem cells and associated with the development and differentiation of urothelial cells. Additionally, we added the Sonic hedgehog (SHH) gene (H), which is expressed in the basement membrane cells of urothelial cells and regulates signals important for cell proliferation during regeneration. These genes were introduced into adult skin fibroblasts (aHDFs) using retroviral vectors, and the cells were then cultured in CnT-Prime medium for a period suitable for cell maintenance (21 days) ( figure 1 ). In FIT or FIH-introduced cells, uroplakin (UPK1b), which is a developmental marker of urothelial cells, was slightly expressed ( figure 2 A). None of the cells formed urothelia...

Embodiment 2

[0302] Characterization of directly transformed urothelial cells (dUCs).

[0303] Cells transformed by the method of Example 1 are represented as directly transformed urothelial cells (dUC). Next, the detailed properties of the cells were investigated. In the analysis over time, cells transformed with F, T, L, and K significantly expressed UPK1b 8 days after gene introduction, and the expression peaked after 15 days ( Figure 8 a). In FTLK-transformed cells, UPK2 mRNA expression started to rise after 11 days and peaked after 21 days. Epithelial colony formation was observed after 11 days and increased thereafter ( Figure 8 b).

[0304] In addition to the expression of UPK1b and UPK2, the expression of E-cadherin (a transmembrane protein expressed in general epithelial cells) and keratin 8 / 18 (a cytoskeleton) was also confirmed. By immunocytochemical analysis, about 42% of cells expressed UPK1b, on the other hand, UPK2-positive, E-cadherin-positive, and keratin 8 / 12-pos...

Embodiment 3

[0307] Pluripotency, liver and small intestine markers were not expressed in dUCs.

[0308] In order to rule out the possibility that fibroblasts are temporarily transformed into iPs cells or endodermal progenitor cells, and thus give rise to urothelial cells, pluripotency markers in cells introduced with F, T, L, K during the transformation were investigated and expression of non-urothelial endoderm markers. As a result, the expression of NANOG, POU5F1, LIN28, and SOX2 genes was not observed in the cells into which F, T, L, and K were introduced during the culture. The expression of NANOG protein was also not observed ( Figure 10 ). Similarly, at any time of observation, F, T, L, and K-introduced cells did not express the mRNA of the small intestinal epithelial cell marker CDX2 and the liver cell marker albumin (ALB) ( Figure 11 ). In addition, the cells into which F, T, L, and K were introduced also did not express the mRNA of the endothelial cell marker CD31 derived...

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Abstract

The invention aims to provide the preparation method of the urinary tract epithelial cells, and the urinary tract epithelial cells can be applied to treatment of urinary system diseases, especially diseases caused by urinary tract epithelial cell injury or diseases caused by urinary tract epithelial cell deficiency or dysfunction. Also provided are urinary tract epithelial cells prepared by the method and a culture medium for inducing (preparing) urinary tract epithelial cells. A urinary tract epithelial cell prepared by a method for inducing a urinary tract epithelial cell can be applied to the treatment of urinary system diseases, the method comprising a step of introducing into a somatic cell of a mammal at least one of the following exogenous factors: FOXA1 (Forkhead box A1) gene or an expression product thereof, TP63 (oncoprotein P63, TP63) gene or an expression product thereof, TP63 (oncoprotein P63, TP63) gene or an expression product thereof, and TP63 (oncoprotein P63, TP63) gene or an expression product thereof. The present invention relates to a recombinant vector comprising: a recombinant vector (1) comprising a recombinant vector (1), a recombinant vector (2), a recombinant vector (3), a recombinant vector (4), a recombinant vector (6), a recombinant vector (7), a recombinant vector (8), a recombinant vector (7), a recombinant vector (8), a recombinant vector (8), a recombinant vector (8), a recombinant vector (8), a recombinant vector (8), a recombinant vector (8), a recombinant vector (8), and a recombinant vector (8).

Description

technical field [0001] The invention mainly relates to a method for inducing urothelial cells, in particular to a method for inducing urothelial cells by direct reprogramming of genes, and a preparation for transforming somatic cells into urothelial cells. Background technique [0002] On the surface of the urinary tract, which consists of the renal pelvis, ureter, bladder, and proximal urethra, urothelial cells make up the urothelium. The urothelium contracts or expands according to the amount of urine stored and acts as a strong barrier to urine exudation or bacterial invasion. Urinary tract diseases such as bladder cancer, Overactive Bladder, Congenital Bladder Abnormalities, Bladder Injury, Interstitial Cystitis, Neurogenic Bladder, Atrophic Bladder, and Loss and / or Function of Urothelial Cells obstacles related. Patients with damaged urothelium are treated by transplanting a section of their own gut or large bowel tissue (eg, surgical techniques to strip the gut epith...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A61K48/00A61P13/00A61P13/02A61P13/10A61P35/00A61P43/00C12N5/10C12N15/12A61K35/22A61K35/76
CPCA61K35/22A61P13/00A61P13/02A61P13/10A61P35/00A61P43/00C12N5/0685C12N2506/1307C12N2501/603C12N2501/604C12N2501/606C12N2501/60C12N2501/33C12N2501/11C12N2740/13043C12N5/0684C12N15/85A61K48/00C12N2510/00C12N2501/999C12N15/86C12N2740/10022C12N2740/10042
Inventor 井上裕太松田修岸田纲郎浮村理关诚
Owner 세루아쿠시아가부시키가이샤
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