Induced malignant stem cells or pre-induction cancer stem cells capable of self-replication outside of an organism, production method for same, and practical application for same

A technology of cancer stem cells and cancer stem cells, applied in the field of inventions involving induced precancerous stem cells or induced malignant stem cells, which can solve problems such as chromosomal abnormalities, cancer cell abnormalities, and deletions

Inactive Publication Date: 2013-01-30
NAT CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, in the cancer cell lines established with ordinary conventional media, there are problems that, due to the marked generation of artificial chromosomal abnormalities (transposition, deletion, etc.), genetic abnormalities (gene mutations), Epigenetic abnormalities that cause gene expression abnormalities are difficult to maintain as they are in the original cancer cells and cancer cells that originally cause cancer in the body
However, there is no report of long-term culture of monoclonal cancer stem cells in vitro self-replication, nor has it been cultured through self-replication and proliferation to obtain sufficient in vitro application of innovative drugs or cancer research. number of reports

Method used

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  • Induced malignant stem cells or pre-induction cancer stem cells capable of self-replication outside of an organism, production method for same, and practical application for same
  • Induced malignant stem cells or pre-induction cancer stem cells capable of self-replication outside of an organism, production method for same, and practical application for same
  • Induced malignant stem cells or pre-induction cancer stem cells capable of self-replication outside of an organism, production method for same, and practical application for same

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Experimental program
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preparation example Construction

[0226] Method for preparing induced cancer stem cells

[0227] In the second aspect of the present invention, a method for preparing the induced cancer stem cells of the present invention is provided, which is selected from the group consisting of (a) somatic cells collected from mammals with tumor suppressor gene mutations, and carcinogenic Preparation of inducible cancer stem cells capable of self-replicating in vitro from non-embryo raw somatic cells collected from mammals with abnormal somatic cells of (a) tumor suppressor gene mutation or (b) cancer-related gene expression increase .

[0228] The method is characterized in that an induction step is performed, which is the basis of the state in which the gene products of the POU5F1 gene, KLF4 gene, and SOX2 gene exist in the raw material somatic cells. As a result, the gene (1) inherent in the raw somatic cell (self-replication-related gene) is expressed, and finally induced into the induced cancer stem cell of the present in...

Embodiment 1

[0309] Example 1. Preparation of retroviral vector

[0310] Using Fugene HD (manufactured by Roche Company (Roche); article number 4709691), the POU5F1-pMXs, KLF4-pMXs, SOX2-pMXs 3-gene retroviral vector plasmids were introduced into the pantropic retroviral vector In the Plat-GP cell of the packaging cell for preparation, a retroviral vector solution is prepared. The ratios of the genes POU5F1-pMXs, KLF4-pMXs, SOX2-pMXs are 4:2:1 in sequence, maintaining the relationship of POU5F1>SOX2. The details are as follows.

[0311]

[0312] POU5F1-pMXs, KLF4-pMXs, SOX2-pMXs are constructed vectors (Table 33 below).

[0313] The amount of each vector is POU5F 1-pMXs 5μg, KLF4-pMXs 2.5μg, SOX2-pMXs1.25μg, Venus-pCS21.25μg, VSV-G-pCMV 5μg, GFP-pMXs1.25μg (produced by CellBiolab), FuGENE HD45μl.

[0314]

[0315] The genes POU5F1-pMXs, KLF4-pMXs, and SOX2-pMXs are constructed vectors (Table 33 below).

[0316] The amount of each vector is POU5F1-pMXs 5μg, KLF4-pMXs 2.5μg, SOX2-pMXs1.25μg, Venus...

Embodiment 2

[0324] Example 2. Preparation of induced malignant stem cells from cells derived from cancer tissues of patients with gastric cancer

[0325] Somatic cells are separated from fresh cancer tissues of human gastric cancer (advanced cancer) patients who have been preserved and transported in the preservation solution for several hours. To the obtained cells derived from the cancer tissues of gastric cancer patients, the retroviral vector prepared in Example 1 that satisfies the relationship of POU5F1>KLF4>SOX2 (the ratio of POU5F1, KLF4, SOX2 is 4:2:1) is added Liquid, introduce genes, and prepare human induced malignant stem cells. The details are as follows.

[0326] Wash a part of fresh gastric cancer (advanced cancer: male, 67-year-old, Japanese) tissue obtained during surgery with Hanks balance solution (without phenol red) (manufactured by Invitrogen; article number 14175-095), and cut to about 0.1 with scissors ~1mm 2 . Furthermore, after washing with Hanks balance solution...

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Abstract

Disclosed are inducible cancer stem cells which are capable of in vitro self-replication and which are useful in research into cancer treatment and cancer-related drug development. Also disclosed are a production method for the above cells, cancer cells induced from the above stem cells, and a practical application of the cells. The disclosed inducible cancer stem cells which are capable of in vitro self-replication are characterised by satisfying requirements (1) and (2): (1) expressing six genes (POU5F1 gene, NANOG gene, SOX2 gene, ZFP42 gene, LIN28 gene, and TERT gene) selected from among a specified gene group; (2) (a) having an abnormality which is either the modification of an endogenous cancer suppression gene, or (b) the up-regulation of an endogenous cancer related gene.

Description

Technical field [0001] The present invention relates to induced precancerous stem cells or induced malignant stem cells, involving abnormalities with endogenous tumor suppressor gene mutations, increased expression of endogenous cancer-related genes, etc., expressing POU5F1 gene, NANOG gene, SOX2 gene, ZFP42 gene, LIN28 Genes, TERT genes and other self-replication-related genes, induced pre-cancer stem cells or induced malignant stem cells capable of self-replication in vitro (the present invention is collectively referred to as "inducible cancer stem cells" hereinafter), methods for preparing these cells, and The application of these cells. technical background [0002] In recent years, by studying embryonic stem cells (also called "ES cells". Hereinafter referred to as "embryonic stem cells"), and by further studying somatic cell clone embryonic stem cells, and the creation of somatic cells cloned animals, so-called so-called somatic Cell cloning is considered to be capable of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A01K67/027A61K39/00C12N15/09C12Q1/02G01N33/15G01N33/50
CPCC12N2501/603C12N15/85A61K39/0011C12N2501/604G01N33/18C12N2510/00G01N33/5011C12N2501/60C12N5/0695G01N33/574C12N2501/602G01N33/5073G01N33/1826A01K2207/12A01K2267/0331A61P35/00A61K39/001157C12N5/10C12N5/0693C12N15/11G01N33/15
Inventor 石川哲也
Owner NAT CANCER CENT
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