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Cultivation system used for generation of chemically induced pluripotent stem cell and chemical reprogramming method using the same

A technology of pluripotent stem cells and culture system, applied in the direction of artificially induced pluripotent cells, non-embryonic pluripotent stem cells, cell culture active agents, etc. The effects of animal-free culture system, reduced cell loss, simplified collection and molecular mechanism analysis of competent stem cells

Inactive Publication Date: 2019-09-10
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Deng Hongkui's research group at Peking University has reported the use of small molecule Forskolin to replace Oct4 in Yamanaka factor to generate chemically induced pluripotent stem cells (Hou, P. et al., Pluripotent Stem Cells Induced From Mouse Somatic Cells by Small-molecule Compounds, Science 341,651-654), but the culture system they reported cannot effectively induce pluripotent stem cells and needs to divide the cells frequently during the culture process, the culture system still needs to be improved (Zhao Y. et al., A XEN-like State Bridges Somatic Cells to Pluripotency During Chemical Reprogramming. Cell, 163, 1678-1691)

Method used

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  • Cultivation system used for generation of chemically induced pluripotent stem cell and chemical reprogramming method using the same
  • Cultivation system used for generation of chemically induced pluripotent stem cell and chemical reprogramming method using the same
  • Cultivation system used for generation of chemically induced pluripotent stem cell and chemical reprogramming method using the same

Examples

Experimental program
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Effect test

Embodiment 1

[0134] Example 1. Generation of pluripotent stem cells in a culture system for generation of chemically induced pluripotent stem cells

[0135] The above mouse embryonic fibroblasts, mouse neoplastic fibroblasts, mouse lung fibroblasts, mouse tail tip fibroblasts and mouse neural stem cells were mixed at a density of 20,000 cells / well (12 wells) or 50,000 Cells / well (6 wells) density, mouse hepatocytes were seeded at 5,000,000 / well (6-well plate) in the following chemical reprogramming medium for chemical induction of pluripotent stem cells. The medium contains iCD1 medium as basal medium and 10 ng / ml BMP4, 10 μM Brdu, 5 μM RepSox, 10 μM Forsklin, 0.1 mM VPA, 0.05 μM AM580, 5 μM EPZ5676, 0.05 μM M DZNep, 5 μM MSGC0946, 50 μg / ml vitamin C, 3 μM CHIR99021 and 10 ng / ml basic fibroblast growth factor. The medium was replaced every day, and after 22 days of culture, the above chemical reprogramming medium was replaced with the following containing N2 (100X), B27 (50X), GlutMax (...

Embodiment 2

[0136] Example 2. Characterization of chemically induced pluripotent stem cells

[0137] The expression levels of endogenous pluripotency genes Oct4, Nanog, Sox2, Esrb, Rex1Dappa5, Sall4 and Cdh1 in ciPSCs were monitored by quantitative RT-PCR technique. Such as figure 2 As shown, the expression levels of these endogenous pluripotency genes were the same as the expression levels of endogenous pluripotency genes Oct4, Nanog, Sox2, Esrb, Rex1Dappa5, Sall4 and Cdh1 detected in mouse embryonic stem cells.

[0138] The transcriptomic properties of ciPSCs were detected by RNA-seq analysis. Such as image 3 As shown, the transcriptomic properties of ciPSCs were identical to those of mouse embryonic stem cells.

[0139] Further, as Figure 4 As shown, protein expression levels of pluripotency genes Oct4, Nanog, Sox2, Esrb and Rex1 in chemically induced pluripotent stem cells were confirmed by immunofluorescence.

Embodiment 3

[0140] Example 3. In vivo differentiation properties of chemically induced pluripotent stem cells in mice

[0141] The ciPSCs obtained in Example 1 were subcutaneously injected into NOD-SCID mice. Such as Figure 5 As shown, ciPSCs formed teratomas in mice and differentiated into cells of the three germ layers (cartilage: mesoderm; muscle: mesoderm; nerve: ectoderm; gut-like epithelium: endoderm). Such as Figure 6 As shown, ciPSCs maintained a normal karyotype during passaging. Inject ciPSCs into mouse blastocysts as Figure 7 As shown, the offspring mice are chimeric mice.

[0142] It can be seen from the above characterization results of Examples 2 and 3 that, according to the method of the present invention, the chemically induced pluripotent stem cells cultured in the culture system of the present invention have full pluripotency. It can be seen that the culture system of the present invention can effectively reprogram somatic cells to generate pluripotent stem cel...

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Abstract

The invention discloses a cultivation system used for generation of a chemically induced pluripotent stem cell, the cultivation system includes a basal culture medium and a composition for promoting chemical reprogramming, wherein the composition for promoting the chemical reprogramming includes a thymine analogue, a cAMP activator, a TGF-[beta] receptor inhibitor, bone morphogenetic protein, a RAreceptor activator, a GSK3 inhibitor, and a basic fibroblast growth factor, and the cultivation system does not comprise serum. In a process of performing the chemical reprogramming of somatic cellsby the method provided by the invention, frequent disc sorting does not require to be performed on cells, compared with an existing cultivation method, operation steps are simplified, cell loss generated in a disc sorting process is reduced, and utilization of the cultivation system provided by the invention does not require usage of the serum, so sequent collection and molecular mechanism analysis of pluripotent stem cells are further simplified, and the system facilitates subsequent establishment of an animal-derived free cultivation system for induced pluripotent stem cells.

Description

technical field [0001] The invention relates to a culture system for chemically inducing pluripotent stem cells and a chemical reprogramming method using the culture system. Background technique [0002] The induction of pluripotent stem cells from somatic cells is a revolutionary discovery in the fields of biology and medicine. For the medical field in particular, the concept of inducing pluripotent stem cells from somatic cells can guide the development of regenerative medicines as patient-specific stem cells for the treatment of degenerative diseases such as Parkinson's disease and Alzheimer's disease . In the biological field, somatic cell reprogramming driven by Yamanaka factors (Oct4, Sox2, Klf4, and Myc), for example, the molecular and cellular mechanisms that control cell fate and its transformation have now been studied in detail. This detailed study of cell fate further reveals the applications of inducing pluripotent stem cells from somatic cells in cancer thera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
CPCC12N5/0696C12N2506/02C12N2506/13C12N2500/40C12N2501/01C12N2501/15C12N2501/155C12N2500/38C12N2501/065C12N2501/115C12N2501/727C12N2501/12C12N2501/165C12N2501/235C12N2506/1307C12N2506/14C12N2501/385C12N1/38
Inventor 裴端卿曹尚涛刘晶余胜勇陈捷凯叶晶李东伟
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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