Induced malignant stem cells

A stem cell and malignant technology, applied in the field of induced malignant stem cells that proliferate, can solve the problems of difficult to maintain abnormality, chromosomal abnormalities, impossible target/compound screening, etc.

Inactive Publication Date: 2014-10-01
NAT CANCER CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, in a cancer cell line constructed using a conventional conventional universal medium without such gene introduction, after long-term culture, artificial (In vitro Artifact) chromosomal abnormalities ( Transposition, deletion, etc.), genomic abnormality (gene mutation), or epigenetic abnormality causing abnormal gene expression (Non-Patent Document 10)
Therefore, there is a problem that it is difficult to suppress the abnormality of In vitro Artifact to a minimum, and it is difficult to maintain abnormalities such as mutations in cancer cells that originally cause cancer or malignancy in vivo in the cells.
[0014] However, so far, cells that can be expanded and cultured corresponding to the analysis results have not been constructed, and it is impossible to use them for functional analysis, XENOGRAFT models, and target / compound screening in drug development

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0307] Example 1: Preparation and induction of malignancy from cells (GC2) derived from cancer tissues of gastric cancer patients stem cell

[0308] From the donor No. 1 human gastric cancer patient (medical information: gastric cancer, female, 67 years old, Japan) stored in a preservation solution (Hank's solution with kanamycin and amphotericin) refrigerated and transported for several hours Human, blood type O, no chemotherapy, no radiotherapy, no immunosuppressive therapy, no history of smoking, no history of drinking, no drug poisoning, no medical treatment, HIV negative, HCV negative, HBV negative, syphilis negative ) Of fresh cancer tissue isolated cells (GC2). Likewise, cells were isolated from fresh non-cancerous tissue (NGC2). To the obtained cells derived from gastric cancer tissue as solid cancer, 4 genes (POU5F1, KLF4, SOX2, c-Myc) Sendai virus vector solution of CytoTune iPS (DV-0301-1) manufactured by DNAVEC Corporation were added to perform gene Introduced, th...

Embodiment 2

[0322] Example 2: Producer induction from cells (CC3) derived from cancer tissues of patients with colorectal cancer Malignant stem cells

[0323] From donor No. 2 human colorectal cancer patients (medical information: sigmoid colon cancer, male, 77) who were refrigerated and transported for several hours in the preservation solution (Hank's solution with kanamycin and amphotericin) Years old, Japanese, blood type A, no chemotherapy, no radiotherapy, no immunosuppressive therapy, no history of smoking, history of drinking beer 1 bottle / day, no drug poisoning, no drug treatment, HIV negative, HCV Negative, HBV-negative, syphilis-negative) fresh cancer tissue isolated cells (CC3). Similarly, cells were isolated from fresh non-cancerous tissue from the same donor (NCC3). To the obtained cells derived from cancer tissues of human colorectal cancer patients, 4 genes (POU5F1, KLF4, SOX2, c-Myc) Sendai virus vector solution of CytoTune iPS (DV-0301-1) manufactured by DNAVEC Co., Ltd....

Embodiment 3

[0333] Example 3: Preparation of retroviral vector

[0334] Using Fugene HD (manufactured by Roche; Cat no.4709691), a retroviral vector plasmid containing the 3 genes of POU5F1-pMXs, KLF4-pMXs, and SOX2-pMXs was introduced into Plat as a packaging cell for the preparation of pan-tropic retroviral vectors -In GP cells, prepare retroviral vector solution. The details are as follows.

[0335]

[0336] POU5F1-pMXs, KLF4-pMXs, SOX2-pMXs were constructed vectors (Table 9).

[0337] The amount of each vector is POU5F1-pMXs 5μg, KLF4-pMXs 2.5μg, SOX2-pMXs 1.25μg, Venus-pCS2 1.25μg, VSV-G-pCMV 5μg, GFP-pMXs1.25μg (manufactured by Cell Biolab), FuGENE HD 45μl .

[0338]

[0339] The genes POU5F1-pMXs, KLF4-pMXs and SOX2-pMXs were constructed vectors (Table 9).

[0340] The amount of each vector was POU5F1-pMXs 5 μg, KLF4-pMXs 2.5 μg, SOX2-pMXs 1.25 μg, Venus-pCS2 1.25 μg, VSV-G-pCMV 5 μg, GFP-pMXs 1.25 μg, and FuGENE HD 45 μg.

[0341]

[0342] The genes POU5F1-pMXs, KLF4-pMXs, SOX2-pMXs were...

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Abstract

The present invention addresses the problem of providing malignant stem cells that can be grown in vitro and are useful in cancer therapy research and drug discovery research for cancer therapy, a method for producing same, cancer cells induced from the cells, and use for the cells. Provided are induced malignant stem cells that can be grown in vitro, the induced malignant stem cells being characterized in (1) having at least one type of abnormality selected from among (a) abnormal methylation (hypermethylation or hypomethylation) of a cancer suppressor gene or cancer-related gene region in the endogenous genome DNA, (b) somatic mutation of a cancer suppressor gene in the endogenous genome DNA or somatic mutation of an endogenous cancer-related gene, (c) abnormal expression (increased expression or decreased / lost expression); of an endogenous cancer gene or endogenous cancer suppressor gene, (d) abnormal expression (increased expression or decreased / lost expression) of non-coding RNA such as endogenous cancer-related micro RNA, (e) abnormal expression (increased expression or decreased / lost expression) of an endogenous cancer-related protein, (f) endogenous cancer-related metabolism abnormality (hypermetabolism or hypometabolism), or (g) abnormal endogenous cancer-related carbohydrate; and (2) expressing the POU5F1 gene, NANOG gene, SOX2 gene, and ZFP42 gene.

Description

Technical field [0001] The present invention relates to induced malignant stem cells, which have genomic abnormalities or epigenetic abnormalities related to cancer, and express POU5F1 gene (also called OCT3 / 4 gene), NANOG gene, SOX2 gene, and ZFP42 gene. , Induced malignant stem cells capable of proliferating in vitro, their production methods, cancer cells induced from these cells, and applications of these cells. Background technique [0002] In recent years, through research on stem cells represented by the creation of cloned animals and embryonic stem cells (also referred to as "ES cells". In this specification, hereinafter referred to as "embryonic stem cells"), it has begun to be considered that epigenetic ( DNA methylation and histone modification) reprogramming (also referred to as "initialization". In this specification, it is referred to as "reprogramming" below). In fact, it is reported that the following experimental results are transplanted into an enucleated egg c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/095C12N5/09C12N5/10
CPCC12N2501/603C12N2501/602C12N2510/00C12N2506/30C12N2501/606G01N33/5073G01N33/5011C12N5/0695C12N2503/00C12N2501/604
Inventor 石川哲也
Owner NAT CANCER CENT
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