Method for preparing induced pluripotent stem cells as well as composition used in method and application of composition

A technology of pluripotent stem cells and compositions, applied in the field of preparation of induced pluripotent stem cells, which can solve problems such as inability to obtain chimera mice

Active Publication Date: 2015-05-20
杭州健崃生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the iPSCs obtained in this experiment are different from embryonic stem cells in terms of gene expression and methylation patterns, and living chimeric mice cannot be obtained.

Method used

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  • Method for preparing induced pluripotent stem cells as well as composition used in method and application of composition
  • Method for preparing induced pluripotent stem cells as well as composition used in method and application of composition
  • Method for preparing induced pluripotent stem cells as well as composition used in method and application of composition

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0058] Preparation method of I.iPSC

[0059] overview

[0060] In general, the present invention provides a method for preparing iPSCs using a composition containing a c-Jun antagonist and a composition containing a c-Jun antagonist for promoting the formation of induced pluripotent stem cells (iPSCs) used in the method, the The composition comprises a c-Jun antagonist, such as a c-Jun antagonist having a bZIP domain but lacking a transactivation domain, antagonizing A compound, antibody or antibody fragment that is c-Jun active.

[0061] In one aspect, the present invention provides a method for preparing induced pluripotent stem cells (iPSCs), the method comprising introducing a composition promoting the formation of induced pluripotent stem cells (iPSCs) into somatic cells, wherein the composition comprises:

[0062] (i) c-Jun antagonist, and a group of factors selected from the following seven groups of factors: (1) Sox2, Klf4 and c-Myc, (2) Klf4 and c-Myc, (3) Oct3 / ...

Embodiment 1

[0258] Example 1. Inhibitory effect of c-Jun on somatic cell reprogramming

[0259] (1) c-Jun plays different roles in somatic cells and stem cells

[0260] In order to study the role of c-Jun in somatic cells and stem cells, mouse embryonic stem cells and mouse embryonic fibroblasts were selected as models, and TRIzol was used to extract the total mRNA in mouse embryonic stem cells and mouse embryonic fibroblasts, Subsequently, qPCR was performed by the procedure described below: cDNA was prepared with ReverTra Ace (Toyobo) and oligo-dT, followed by qPCR (Takara) analysis with Premix Ex Taq.

[0261] Figure 1A The expression levels of c-Jun in mouse embryonic stem cells, induced pluripotent stem cells and mouse embryonic fibroblasts are compared with Oct4 and Nanong. Such as Figure 1AAs shown, the expression of c-Jun is lower in mouse embryonic stem cells and induced pluripotent stem cells, but higher in mouse embryonic fibroblasts.

[0262] In order to further verify t...

Embodiment 2

[0325] Example 2. Role of the bZIP domain in c-Jun

[0326] 1. Effects of c-Jun domains on somatic cell reprogramming

[0327] The JNK-phosphorylation site (Ser63 / Ser73) mutant of c-Jun and the truncated c-Jun located at amino acid 170 to amino acid 334 with a bZIP domain but lacking a transactivation domain were constructed by molecular cloning methods. Jun (c-JunDN). The amino acid sequence of wild-type c-JunWT is the following SEQ ID NO.1; the amino acid sequence of c-JunDN is SEQ ID NO.2.

[0328] Amino acid sequence (SEQ ID NO.1) of wild-type c-JunWT (ie, full-length c-Jun):

[0329] mtakmettfy ddalnasflq sesgaygysn pkilkqsmtl nladpvgslk phlraknsdl 60

[0330] ltspdvgllk laspelerli iqssnghitt tptptqflcp knvtdeqegf aegfvralae 120

[0331] lhsqntlpsv tsaaqpvsga gmvapavasv agaggggggys aslhseppvy anlsnfnpga 180

[0332] lssgggapsy gaaglafpsq pqqqqqppqp phhlpqqipv qhprlqalke epqtvpempg 240

[0333] etpplspidm esqerikaer krmrnriaas kcrkrkleri arleekvktl kaqnselast 300 ...

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Abstract

The invention provides a method for preparing induced pluripotent stem cells and a composition used in the method. The method comprises the following step: introducing a composition of promoting induced pluripotent stem cells to form into a body cell, wherein the composition comprises (i) a c-Jun antagonist, and a group of factors selected from the following seven groups of factors (1) Sox2, Klf-4 and c-Myc, (2) Klf4 and c-Myc, (3) Oct3 / 4, Klf4 and c-Myc, (4) Sox2, Nanog and Lin28, (5) Oct3 / 4, Nanog and Lin28, (6) Oct3 / 4, Klf and Sox2 and (7) Klf4 and Sox2; or (ii) a c-Jun antagonist, Jhdm1b and Id1, and at least one of Glis1, Sall4 or Lrh1; or (iii) a c-Jun antagonist, Jhdm1b and Id1, and at least one of Oct4, Klf4, Sox2, Lin28, Esrrb, Lef1, Utf1 or miRNA C. Through the method disclosed by the invention, the induced pluripotent stem cells can be successfully prepared; the obtained induced pluripotent stem cells are good in quality; and no abnormal chromosome is generated.

Description

technical field [0001] The invention relates to a method for preparing induced pluripotent stem cells, a composition for promoting the formation of induced pluripotent stem cells (iPSC) and uses thereof. Background technique [0002] Stem cells are a type of cells capable of self-renewal through cell mitosis, and can differentiate into various specialized cells under specific conditions. This self-renewal capacity of stem cells is used as the basis for the cellular differentiation and specialization required for organ and tissue development. Recent evidence shows that stem cells can be used for tissue reconstruction and restoration of physiological function and tissue function. Therefore, stem cells have the potential to be used in the treatment of a variety of diseases and injuries, including nervous system trauma, malignancy, genetic disorders, hemoglobinopathy, and immunodeficiency. Stem cell transplantation therapy is an important direction of regenerative medicine res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
CPCC12N5/0696C12N2501/60C12N2501/602C12N2501/603C12N2501/604C12N2501/605C12N2501/606C12N2501/608C12N2506/02
Inventor 裴端卿刘晶陈捷凯彭梅秀
Owner 杭州健崃生物科技有限公司
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