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Composition and method for generating induced pluripotent stem cells using the same

A technique for embryonic stem cells and cells, applied in the field of compositions and the generation of induced totipotent stem cells therewith

Inactive Publication Date: 2011-11-16
STEMLAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] However, there is no method known in the art for inducing dedifferentiation and forming totipotent embryonic stem cell-like cells by introducing the Bmi1 gene and treating it with a low-molecular-weight substance

Method used

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  • Composition and method for generating induced pluripotent stem cells using the same
  • Composition and method for generating induced pluripotent stem cells using the same
  • Composition and method for generating induced pluripotent stem cells using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Culture of Mouse Somatic Cells and Introduction of Bmi1 Gene

[0098] Mouse somatic cells, specifically mouse embryonic fibroblasts, are used to generate embryonic stem cell-like cells. Embryos were obtained from CF1 mice at an embryonic age of 13.5 days. In tissue culture flasks, cells were incubated in DMEM (high glucose, w / o acetone) supplemented with 10% FBS (fetal bovine serum), 0.1 mM non-essential amino acids, 1% penicillin / streptomycin, and 0.1 mM β-mercaptoethanol Sodium acid), after that, the third-generation fibroblasts were cultured in 2×10 5 Density of cells / well seeded in 6-well plates.

[0099] For gene transfer, retroviral particles were prepared from the PT67 packaging cell line. In this background, pBabe puro Bmi1 (from Dr. G.P. Dimri, Evanston Northwestern Healthcare Research Institute, Feinberg School of Medicine, Northwestern University, Evanston, IL 60201, USA) was transfected into the PT67 packaging cell line with the aid of Turbofect (Fermenta...

Embodiment 2

[0102] Reprogramming of Bmi1-transduced somatic cells into blastoid-like cells under specific culture conditions

[0103] When cultured under EGF-free conditions (DMEM / F12+B27+N2+1% penicillin / streptomycin+20ng / ml bFGF) for neural stem cells, the mice in which the Bmi1 gene was introduced with retrovirus The cells are reprogrammed to form neurosphere-like cells that subsequently develop into blastoid-like cells ( Figure 1A ). By RT-PCR analysis, it was found that these cells express the genes of blast cells ( Figure 1B ).

Embodiment 3

[0105] Selection of Oct4-positive cells using Oct4-promoter-GFP, reprogramming into iPS cells by treatment with low molecular weight substances PD0325901 and CHIR99021, and characterization of embryonic stem cells

[0106] 1) Selection of Oct4-positive cells using Oct4-promoter-GFP

[0107] To compensate for the fact that it was observed that the dedifferentiated stem cells established in Example 2 did not form chimeras, importing (refer to Nature 448, 318-324), and then use GFP to select Oct4 positive cells.

[0108] Selected cells can be further developed into cells capable of forming chimeras. However, they cannot be considered fully reprogrammed cells because they differ from embryonic stem cells in terms of gene expression.

[0109] 2) Reprogramming into iPS cells by treatment with low molecular weight substances PD0325901 and CHIR99021

[0110] Oct4-positive cells were treated with 0.5 μM PD0325901 and 3 μM CHIR99021, and then induced to reprogram under the culture c...

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Abstract

The present invention relates to a composition and a method for generating induced pluripotent stem cells using the same Provided is a composition for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising: a) a Bmi1 (B cell-specific Moloney murine leukemia virus integration site 1) protein or a nucleic acid molecule encoding the Bmi1 protein; and b) at least one low molecular weight substance selected from the group consisting of a set of a MEK / ERK (mitogen-activated protein kinase / extracellular regulated kinase) inhibitor and a GSK (glycogen synthase kinase) inhibitor, a set of a G9a HMTase (G9a histone methyltransferase) inhibitor and a DMNT (DNA methyltransferase) inhibitor, and a histone deacetylase inhibitor. Also, a method is provided for reprogramming somatic cells to generate embryonic stem cell-like cells using the composition.

Description

technical field [0001] The present invention relates to a composition for reprogramming somatic cells to produce embryonic stem cell-like cells, and a method for producing embryonic stem cell-like cells using the composition, the composition comprising Bmi1 and a low molecular weight substance. More specifically, the present invention relates to a composition for reprogramming somatic cells to produce embryonic stem cell-like cells by introducing Bmi1 into somatic cells and treating the cells with low-molecular-weight substances comprising MEK inhibitors and GSK inhibitors, and using the A method for producing embryonic stem cell-like cells from the composition. Background technique [0002] Unlike ordinary somatic cells, stem cells possess self-renewal (ie, the ability to remain undifferentiated through multiple cycles of cell division) and potency (ie, the ability to differentiate into specialized cell types under suitable conditions). Potency refers specifically to the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
CPCC12N2501/998C12N2501/60C12N2501/065C12N2501/70C12N2799/027C12N5/0696C12N15/873C12N2501/115C12N2501/727
Inventor 刘承权文哉喜金俊成尹炳善李重翰全恩暻许峻硕金智儇黄志惠权秀贤
Owner STEMLAB
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