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Method for inducing pluripotent stem cells and pluripotent stem cells prepared by said method

Inactive Publication Date: 2016-07-28
AMOREPACIFIC CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present disclosure describes a method of making stem cells using an extract of plant stem cells or an induced pluripotent plant stem cell. This method is safe and can be used on cells of any species, including humans. It is free from ethical concerns because it uses a plant-derived stem cell extract. The technical effect of this method is the ability to prepare stem cells in a safe and efficient manner.

Problems solved by technology

However, the pluripotent embryonic stem cells raise serious religious and ethical concerns implicated with the destruction of embryos during preparation thereof.
In addition, since they are derived from limited embryos, immune rejection due to lack of immunocompatability between individuals cannot be avoided.
The somatic cell nuclear transfer is very inefficient and there is an ethical question in that it requires eggs in large quantities.
The fusion with ES cells has a serious problem in terms of cell stability because the induced cells additionally have two pairs of genes.
The reprogramming by defined factors, which has been reported most recently, involves the serious problem of carcinogenesis because it uses oncogene-containing viruses.
First, a large quantity (20 mg or more) of iPSC extract is necessary for this method.
For this reason, induction of dedifferentiation using an extract of human-derived dedifferentiated stem cells, which is costly and requires much labor, has not been successful.
For example, to prepare human-derived dedifferentiated stem cells for obtaining 20 mg of extract, an expert has to work hard for at least 3 months, which is very costly.
Second, when somatic cells to be induced are treated with an extract of animal stem cells or dedifferentiated stem cells thereof, if the cells survive in the extract without being completely destroyed, it is not easy to distinguish the dedifferentiation-induced cells from the surviving dedifferentiated stem cells and analysis of genomic DNA is necessary, which is costly and time-consuming.
Third, since preparation of human-derived dedifferentiated stem cells using proteins has been hardly successful, an extract of human-derived dedifferentiated stem cells prepared using viruses has to be used.
Because the resulting cells may contain oncogenic substances derived from the viruses, there may be difficulty in clinical application.

Method used

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  • Method for inducing pluripotent stem cells and pluripotent stem cells prepared by said method
  • Method for inducing pluripotent stem cells and pluripotent stem cells prepared by said method
  • Method for inducing pluripotent stem cells and pluripotent stem cells prepared by said method

Examples

Experimental program
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Effect test

example 2

Preparation of Pluripotent Cells Such as Embryonic Stem Cells by Culturing Extract-Injected Cells

[0160]The extract-injected cells were incubated in a normal cell culture medium wherein DMEM (Dulbecco's modified Eagle's medium) was supplemented with 10% FBS, 50 U / mL penicillin and 50 mg / mL streptomycin in an incubator maintained at 37° C. and 5% CO2. The adult-derived cells (human-derived dermal fibroblasts) into which the plant stem cell extract (callus powder) was injected were cultured on a dish coated with 0.1% gelatin. The medium was replaced after the first two days. After culturing for 10 days while replacing the medium every day, the cells were transferred to a feeder cell (STO cell) layer treated with mitomycin C (MMC) at a ratio of 1:2. Then, the cells were transferred to a new feeder cell layer with 7-day intervals while replacing DMEM (Dulbecco's modified Eagle's medium) / F12 supplemented with 20% KSR (knockout serum replacement), 2 mM L-glutamine, 0.1 mM nonessential amin...

example 3

Characterization of Induced Pluripotent Stem Cells (Gene Expression Analysis)

[0163]The cultured cells were recovered and total RNA was separated by using the TRIzol reagent (Invitrogen). After synthesizing cDNA through reverse transcription polymerase chain reaction (RT-PCR), PCR was conducted using primers specific for the Nanog and Oct3 / 4 genes and the GAPDH gene as a control gene. The expression of these genes was analyzed by electrophoresing the PCR product on an agarose gel. The result is shown in FIG. 7.

[0164]As seen from FIG. 7, the pluripotent stem cells (hiPS) induced by the method of the present disclosure showed expression of the Nanog and Oct3 / 4 genes, which are characteristic of embryonic stem cells (hES).

example 4

Preparation of Sequoia (Sequoiadendron giganteum) Callus Extract Containing Shikimic Acid

[0165]20 mg of the sequoia callus powder of Example 1 was dissolved in 1 mL of a DMSO solvent. Similarly, 1 g of the sequoia callus powder was dissolved in 10 mL of a mixture solvent of BG and EtOH to prepare a sequoia callus extract containing shikimic acid. The prepared extracts were used as samples in the following test example.

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Abstract

The present disclosure relates to a method for inducing pluripotent stem cells by inducing reprogramming and / or dedifferentiation of differentiated adult cells using shikimic acid, a plant extract or plant stem cells containing shikimic acid and an extract of dedifferentiated stem cells (callus), pluripotent stem cells prepared by the method and a composition containing the pluripotent stem cells. In accordance with the present disclosure, ethical concerns implicated with the use of eggs to prepare pluripotent stem cells such as embryonic stem cell can be resolved. And, because the plant stem cell extract unharmful to human is used, pluripotent stem cells with proven safety can be prepared and they may be used to develop immunocompatible cell therapy agents suited for individuals. In addition, by pluripotent stem cells from individuals having diseases, the present disclosure will be very useful in studying the cause of diseases and devolving therapeutic strategy.

Description

TECHNICAL FIELD[0001]The present disclosure relates to a method for inducing pluripotent stem cells by reprogramming and / or dedifferentiating a differentiated adult cells, pluripotent stem cells prepared by the method and a composition containing the pluripotent stem cells.[0002]The present disclosure also relates to a pharmaceutical composition or a cosmetic composition containing pluripotent stem cells.[0003]The present disclosure also relates to a composition for activating stem cells, proliferating skin cells, regenerating skin or anti-aging.BACKGROUND ART[0004]Stem cells are undifferentiated cells that can differentiate into various types of cells constituting biological tissues and can be obtained from the tissues of embryos, fetuses and adults. Among the different cell types of the stem cell, pluripotent stem cells refer to the stem cells that can differentiate into any of the three germ layers, i.e., the endoderm, mesoderm and ectoderm.[0005]The stem cells can be classified ...

Claims

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Application Information

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IPC IPC(8): C12N5/074A61K36/14A61Q19/08A61K31/191A61K8/365
CPCC12N5/0696A61K31/191A61K8/365A61Q19/08A61K36/14A23V2002/00C12N2506/02C12N2502/13A61K2800/74C12N2501/603C12N2501/73A23L1/3002A61K8/368A61K35/28A61K2800/91A23L33/10A23L33/105A61K8/9761A61K8/9767A61K8/9771A61K8/9789A61K8/9794A61P17/00A61P39/06A61P43/00C12N2500/14C12N2500/76C12N2501/115C12N2502/02C12N2506/1307C12N2533/54A61K35/12C07C62/32C12N5/00C12N5/0607A23L2/52A61K8/0204A61K8/0216A61K8/0225A61K8/06A61K8/11A61K8/981A61K9/0019A61K9/0056A61K9/0095A61K9/14A61K9/20A61K9/48A61K2800/78A61K2800/92A61Q19/00
Inventor KIM, AH REUMKIM, SU NAPARK, WON SEOKKWON, YOO WOOKPARK, YOUNG BAEKIM, HYO SOOPAEK, JAE SEUNG
Owner AMOREPACIFIC CORP
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