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 ce

Method used

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  • Undifferentiated cell detection method
  • Undifferentiated cell detection method
  • Undifferentiated cell detection method

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0085][Example 1]

[0086]Materials and Method

[0087]●iPSC

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

[0089]●Mesoderm differentiation

[0090]Place undifferentiated iPSCs on a laminin-coated plate at 1~2x10^4 cells / cm in the presence of ROCK inhibitor (Y-27632)2The cells were inoculated at the density of DMEM F12+B27+CHIR+BMP4 for 4 days, and the cells were used as Mesoderm cells.

[0091]●Ectoderm differentiation

[0092]Place undifferentiated iPSCs on a laminin-coated plate at 1x10^5 cells / cm in the presence of ROCK inhibitor (Y-27632)2The cells were inoculated in the presence of DMEM F12+KSR+2-ME+EGF+2bFGF+SB431542+BIO for 7 days, and the cells were used as ectoderm cells (Ectoderm). Or in the presence of KBM-NSC+B27+bFGF+hLIF+CHIR99021+SB431542 for 7 days, ectoderm cells (Ectoderm) can also be obtained.

[0093]●Undifferentiated residue

[0094]In order to quantify the remaining undifferentiated cells in the cells differentiate...

Example Embodiment

[0155][Example 2]

[0156]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, antibodies can be used. Detection of specifically expressed genes, etc.), even if the expression of the marker gene of the present invention is not directly detected, residual undifferentiated cells can be detected.

Example Embodiment

[0157][Example 3]

[0158]For multiple iPS cell lines, the expression of the marker gene of the present invention is evaluated for target cells and cells induced to differentiate using a three germ layer differentiation kit, etc., by selecting iPS cell lines with low expression of the marker gene, 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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