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Undifferentiated cell detection method

A differentiated cell, undifferentiated technology, applied in biochemical equipment and methods, analytical materials, measuring devices, etc., can solve the problem of inability to use iPS cells, and achieve the effect of reducing the risk of cancer.

Pending Publication Date: 2021-01-22
PUBLIC UNIV CORP YOKOHAMA CITY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, there is a problem that LIN28 reported so far (Non-Patent Document 1) has low expression in differentiated cells of internal organs / tissues (retinal pigment epithelial cells), and on the other hand, in differentiated cells such as hepatocytes, Since expression can be observed in cells induced to differentiate from iPS cells, it cannot be used for detection of residual / incorporation of undifferentiated iPS cells targeting various differentiated cells

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0086] Materials and methods

[0087] ● iPSCs

[0088] Those provided by Kyoto University and Tokyo University (TkDA3-4, 1231A3, 1383D2, 1383D6 and Ff01) were used.

[0089] ●Mesoderm differentiation

[0090] Undifferentiated iPSCs were plated on laminin-coated plates at 1-2x10^4 cells / cm in the presence of ROCK inhibitor (Y-27632). 2 The density was inoculated, cultured in the presence of DMEM F12+B27+CHIR+BMP4 for 4 days, and the cells were regarded as mesoderm cells (Mesoderm).

[0091] ●Ectoderm differentiation

[0092] Undifferentiated iPSCs were plated on laminin-coated dishes at 1x10^5 cells / cm in the presence of ROCK inhibitor (Y-27632) 2 The density was inoculated, cultured in the presence of DMEM F12+KSR+2-ME+EGF+2bFGF+SB431542+BIO for 7 days, and the cells were regarded as ectoderm cells (Ectoderm). Or cultured in the presence of KBM-NSC+B27+bFGF+hLIF+CHIR99021+SB431542 for 7 days to obtain ectoderm cells (Ectoderm).

[0093] ●Undifferentiated remnants

[009...

Embodiment 2

[0156] Antibodies, etc. can be used by introducing reporter protein genes (fluorescent protein genes such as luciferase gene, GFP gene, etc., or mouse CD4 gene, etc.) under the control of the promoter region involved in the expression control of the marker gene of the present invention. detection of specifically expressed genes, etc.), it is possible to detect residual undifferentiated cells without directly detecting the expression of the marker gene of the present invention.

Embodiment 3

[0158] For a plurality of iPS cell lines, target cells and cells induced to differentiate using a 3 germ layer differentiation kit etc. are evaluated for the expression of the marker gene of the present invention, and by selecting an iPS cell line with a lower expression of the marker gene, thereby It is possible to select undifferentiated cell lines with high safety.

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Abstract

Provided is a marker gene capable of detecting the persistence / admixture of undifferentiated cells in a differentiated cell population. Undifferentiated cells present in a differentiated cell population are detected by using at least one gene selected from the group consisting of ESRG, VSNL1, THY1, SFRP2, SPP1, USP44, and CNMD as an undifferentiation marker. A method for detecting undifferentiatedcells, a method of use as an undifferentiation marker, and an undifferentiated cell detection kit. Also provided is a method for selecting an undifferentiated cell line.

Description

technical field [0001] The present invention relates to methods for the detection of undifferentiated cells. Background technique [0002] From the viewpoint of cancer risk, it is extremely important to detect and evaluate the residual / incorporation of undifferentiated iPS cells in differentiated cell populations used in regenerative medicine. So far, as a method for detecting and evaluating the residual / incorporation of undifferentiated iPS cells, a method for detecting iPS cell-specific genes based on quantitative PCR (qPCR) has been reported (Non-Patent Document 1), which uses differentiated cells to utilize undifferentiated cells. The method of re-culturing under the culture maintenance conditions (Non-Patent Document 2). [0003] In the prior art, the re-cultivation method has the advantage of high accuracy because clones are formed from mixed undifferentiated iPS cells, but on the other hand, it takes more than one week to detect, so the method using quantitative PCR ...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/6834C12Q1/6837C12Q1/6841C12Q1/686C12Q1/6869C12Q1/6881C12Q1/6897G01N33/50G01N33/53G01N33/68C12N15/09
CPCC12Q1/6881C12Q2600/158C12Q1/6876C12Q1/6897G01N33/5005G01N33/6896
Inventor 谷口英树关根圭辅
Owner PUBLIC UNIV CORP YOKOHAMA CITY UNIV
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