Method of using annulet free carrier to efficiently reprogramming blood cells to generate iPSC

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method of using annulet free carrier to efficiently reprogramming blood cells to generate iPSC. The method includes following steps: S1, extracting a monocyte from a human blood cell, and selectively culturing the monocyte through an amplification culture medium to obtain a red blood cell ancestral cell; S2, guiding the annulet free carrier expressing exogenous reprogramming factor into the red blood cell ancestral cell obtained in the S1; S3, culturing the red blood cell ancestral cell obtained in the S2 and containing the annulet free carrier expressing the exogenous reprogramming factor through a multipotent stem cell induced culture medium, and inducing in a feed-layer-free system into a reprogramming intermediate-state cell; S4, after complete inducing is finished, replacing the multipotent stem cell induced culture medium in the S3 with a multipotent stem cell culture medium to maintain culturing, and obtaining a cell with expression of the exogenous reprogramming factor expression disappearing and expression of endogenous multipotent genes POU5F1, NANOG, TRA-1-60 and TRA-1-81 activated, namely iPSC.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently reprogramming blood cells to generate iPSCs with a free microcircle carrier. Background technique [0002] In 2006, Takahashi and Yamanaka used retroviral vectors to transfer four transcription factors OSKM (octamer-binding transcription factor 4, Oct4), sex determining region Y box protein 2 (sex determining region Y box protein 2, Sox2), Kuppel-like factor 4 (kruppel-likefactor 4, Klf4) and C-myelocytomatosis oncogene (c-Myc) were transferred into mouse fibroblasts to obtain induced pluripotent stem cells (induced Pluripotent Stem Cell, iPSC). Signed that iPSCs are very similar to embryonic stem cells (ESCs) in terms of biological characteristics, and effectively avoid problems such as ethical restrictions and difficulties in obtaining materials, and have great potential in disease models, drug screening, developmental biology research, and cel...

AI Technical Summary

Problems solved by technology

[0011] Since the viral vectors used to transduce foreign genes into donor cells will have a certain impact on them during the reprogramming process, there are gene rearrangements, karyotype abnormalities, epigenetic abnormalities, etc. in the clones screened by this method phenomenon, even with a high risk of carcinogenicity

In 2008, Okita et al. obtained mouse iPSCs through multiple conventional plasmid transfection methods, but the operation was cumbersome and the reprogramming efficiency was low

[0022] In 2010, Jia et al. used the Minicircle vector to obtain non-integrated iPSCs from human adipose-derived mesenchymal stem cells, but multiple transfections were required, the operation was cumbersome, and the efficiency was limited.

However, partially reprogrammed iPSCs cannot silence the expression of exogenous viral transgenes, which increases the risk of tumorigenicity and greatly limits its clinical application.

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