A kind of non-viral ipscs induction method and its induction composition, kit and ipscs obtained therefrom

A non-viral, small-molecule compound technology, applied in the direction of artificially induced pluripotent cells, viruses/phages, biochemical equipment and methods, etc., can solve the problems of iPSCs cell tumorigenesis, achieve high applicability, and shorten the induction culture time , the effect of reducing the risk of clinical application

Active Publication Date: 2019-08-09
FUTURE HOMO SAPIENS INST OF REGENERATIVE MEDICINE CO LTD (FHSR)
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it has been reported that the widely used episomal plasmid system for somatic cell reprogramming contains at least one of many high-risk oncogenes or factors, such as c-MYC, SV40-LT, TP53 inhibitors and other oncogenic factors. iPSCs obtained from these high-risk factors may have risks such as cell tumorigenesis

Method used

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  • A kind of non-viral ipscs induction method and its induction composition, kit and ipscs obtained therefrom
  • A kind of non-viral ipscs induction method and its induction composition, kit and ipscs obtained therefrom
  • A kind of non-viral ipscs induction method and its induction composition, kit and ipscs obtained therefrom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] This embodiment 1 provides a non-viral iPSCs induction method, comprising the following steps:

[0147] 1) constructing DNA sequences expressing reprogramming factors OCT4, SOX2, GLIS1, KLF4, L-MYC and hsa-miR-302s into episomal plasmids to obtain recombinant plasmids;

[0148] Wherein, the hsa-miR-302s is hsa-miR-302cluster, and its sequence is shown in SEQ ID No.12;

[0149] Among them, the reprogramming factors OCT4 and GLIS1 are connected through the P2A co-expression element and use the EF-1α promoter to initiate transcription, and KLF4 and SOX2 are connected through the P2A co-expression element and use the EF-1α promoter to initiate transcription, and will contain OCT4, The DNA sequences of the four genes GLIS1, KLF4, and SOX2 were jointly constructed into an episomal plasmid; the reprogramming factors L-MYC and hsa-miR-302s were transcribed through the EF-1α promoter and the CMV promoter, respectively, and their The DNA sequence is constructed into an episomal ...

Embodiment 2

[0160] This example is based on Example 1. During the induction culture process in step 2), a small molecular compound is added to stimulate the induced reprogramming process.

[0161] A method for inducing non-viral iPSCs, comprising the following steps:

[0162] 1) constructing DNA sequences expressing reprogramming factors OCT4, SOX2, GLIS1, KLF4, L-MYC and hsa-miR-302s into episomal plasmids to obtain recombinant plasmids;

[0163] Wherein, the hsa-miR-302s is hsa-miR-302cluster, and its sequence is shown in SEQ ID No.12;

[0164] Among them, the reprogramming factors OCT4 and GLIS1 are connected through the P2A co-expression element and use the EF-1α promoter to initiate transcription, and KLF4 and SOX2 are connected through the P2A co-expression element and use the EF-1α promoter to initiate transcription, and will contain OCT4, The DNA sequences of the four genes GLIS1, KLF4, and SOX2 were jointly constructed into an episomal plasmid; the reprogramming factors L-MYC an...

Embodiment 3

[0169] This example provides a non-viral iPSCs induction method. In this method, in addition to the reprogramming factors OCT4, SOX2, GLIS1, KLF4, L-MYC and hsa-miR-302s, the high risk factors c-MYC and SV40 are also studied -The effect of LT or TP53 on iPSCs to study the effect of different reprogramming factors on the karyotype of iPSCs.

[0170] Among them, c-MYC, SV40-LT or TP53 shRNA were respectively constructed into episomal plasmids, or TP53 inhibitor TP53 siRNA was directly transfected into somatic cells, or Pifithrin-μ or Pifithrin-αhydrobromide was directly added to somatic cells.

[0171] A method for inducing non-viral iPSCs, comprising the following steps:

[0172] 1) The DNA sequences expressing reprogramming factors OCT4, SOX2, GLIS1, KLF4, L-MYC and hsa-miR-302s were constructed into episomal plasmids as the control group, that is, group 1; on the basis of the control group, the Add high-risk factors c-MYC, SV40-LT or TP53 shRNA to the episomal plasmid as sho...

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Abstract

The present invention discloses a non-viral iPSCs induction method thereof and inducing compositions thereof, kits and iPSCs thereof. Specifically, the induction method comprises the following steps: 1) the DNA sequence expressing the reprogramming factors POU5F1, SOX2, GLIS1, KLF4, MYCL and hsa-miR-302s is constructed into an additional plasmid to obtain a recombinant plasmid; 2) Introduce the recombinant plasmid obtained from step 1) into human somatic cells for induction culture to obtain iPSCs. This method reduces the clinical applicability risk of iPSCs by using a combination of high-safety reprogramming factors without adding high-risk reprogramming factors such as c-MYC, SV40-LT, TP53 inhibitors, etc. This method has high applicability.

Description

technical field [0001] The invention relates to a non-viral iPSCs induction method, an induction composition, a kit and the obtained iPSCs, belonging to the fields of bioengineering technology and regenerative medicine. Background technique [0002] Classical stem cell biology generally believes that cell differentiation is unidirectional and irreversible. In 1962, British scientist John B. Gurdon transplanted the nucleus of frog intestinal cells into an enucleated frog egg and developed it into a normal tadpole. A similar technique, programmed to a pluripotent cell state with an early stage of embryonic development, has also given rise to various cloned mammals. Forty years after Gurdon's results were reported, Japanese scientist Shinya Yamanaka successfully used four transcription factors (OCT4 (standard gene name: POU5F1), SOX2, KLF4, c-MYC, OSKM) carried by retroviruses in 2006 to successfully Mouse fibroblasts were reprogrammed into induced pluripotent stem cells (iPSC...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10
CPCC12N5/0696C12N15/85C12N2501/065C12N2501/727C12N2501/65C12N2501/71C12N2501/60C12N2501/602C12N2501/604C12N2501/606C12N2800/107C12N2800/108C12N2820/60C12N2501/999C12N2510/00
Inventor 王淋立陈月花宋立兵莫健
Owner FUTURE HOMO SAPIENS INST OF REGENERATIVE MEDICINE CO LTD (FHSR)
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