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Method for establishing induced pluripotent stem cells from human amniotic epithelial cells

A technology of pluripotent stem cells and epithelial cells, applied in the field of reprogramming human amniotic epithelial cells into induced pluripotent stem cells, which can solve the problems of cumbersome process and achieve the effect of sharp edges and large nuclear-to-cytoplasmic ratio

Pending Publication Date: 2021-02-26
TONGJI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can quickly obtain iPSCs, the process is a bit cumbersome and introduces animal-derived substances

Method used

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  • Method for establishing induced pluripotent stem cells from human amniotic epithelial cells
  • Method for establishing induced pluripotent stem cells from human amniotic epithelial cells
  • Method for establishing induced pluripotent stem cells from human amniotic epithelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Reprogramming steps and effects of human amniotic epithelial cells

[0047] Steps of reprogramming human amniotic epithelial cells into hAE-iPSCs in the present invention ( figure 1 ) and the effects are as follows: Day -2: planting cells

[0048] Take 5×10 4 Cells per square centimeter The hAECs cultured in vitro within two passages were plated in a 6-well culture plate, that is, each well of the 6-well culture plate was planted with 4.8×10 5 hAECs.

[0049] Day 0: Transfection

[0050] Take one well of a 6-well culture plate as an example

[0051] 1) Two hours before transfection, replace the cells with fresh human amniotic epithelial cell medium hAECM;

[0052] 2) Dilute the plasmids: Take 1 μg each of the plasmids pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, and pCXLE-hUL and mix them with 100 μL DMEM;

[0053] 3) Add 3 μL Neofect directly to the DNA diluent TM Gently mix the transfection reagent, let it stand at room temperature for 20 minutes, and the transf...

Embodiment 2

[0067] Embodiment 2 Reprogramming steps and effects of human dermal fibroblasts

[0068] The reprogramming steps and effects of the above method in human dermal fibroblasts are as follows:

[0069] Day -2: Seeding cells

[0070] Take 5×10 4 The density of cells per square centimeter spread the human dermal fibroblasts cultured in vitro within four generations in a 6-well culture plate, that is, each well of the 6-well culture plate was 4.8×10 5 Individual dermal fibroblasts.

[0071] Before transfection, it was observed that the morphology and viability of human dermal fibroblasts in the culture plate were normal ( image 3 a).

[0072] Day 0: Transfection

[0073] Take one well of a 6-well culture plate as an example

[0074] 1) Two hours before transfection, replace the cells with fresh human fibroblast culture medium;

[0075] 2) Dilute the plasmids: Take 1 μg each of the plasmids pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, and pCXLE-hUL and mix them with 100 μL DMEM;

[0076...

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Abstract

The invention provides a novel method for reprogramming human amniotic epithelial cells into induced pluripotent stem cells, comprises following steps: introducing a plurality of oriP / EBNA1 free plasmid vectors which carrying six reprogramming factors including five transgenes such as OCT4, SOX2, KLF4, LIN28 and L-MYC and one P53 gene shRNA into hAECs by using a non-lipid cation transfection reagent NeofaectTM. The method is simple in process, convenient to operate and low in cost, and the free plasmid vector is a non-gene integrated reprogramming method, so that tumorigenicity is reduced; thewhole reprogramming process is short in time consumption and high in efficiency; the obtained hAE-iPSCs clone is subjected to enlarged culture in a TeSRTM-E8TM-human recombinant vitronectin system without animal origin, which lays a foundation for clinical application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for reprogramming human amniotic epithelial cells into induced pluripotent stem cells. Background technique [0002] Induced pluripotent stem cells (iPSCs) technology is a technology that obtains self-renewal ability and multi-lineage differentiation potential by introducing exogenous genes or gene products to dedifferentiate somatic cells. iPSCs have great application prospects in cell replacement therapy, gene therapy, disease models, developmental models, and drug screening. Patient-specific or disease-specific iPSCs can help us better understand and treat diseases. At present, iPSCs have been used in the treatment of some diseases, including genetic diseases, degenerative diseases and so on. The advantage of iPSCs is that they can be generated from adult somatic cells without ethical issues, and patient-specific iPSCs have low immunogenicity for autologous ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N5/074
CPCC12N15/85C12N5/0696C12N2506/09C12N2501/603C12N2501/602C12N2501/604C12N2501/608C12N2501/999C12N2501/606C12N2506/025C12N2533/50
Inventor 陆建峰张传宇蒙都曹立宁
Owner TONGJI UNIV
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