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Media and culture methods for ESC differentiation of ectodermal precursor cells

A technology of differentiation medium and precursor cells, which is applied in the field of medium for the differentiation of ESCs to ectoderm precursor cells, can solve the problems of complex components of differentiation medium, unfavorable large-scale use, complicated operation process, etc., and achieve the benefit of large-scale Production and use, low cost, high differentiation efficiency

Active Publication Date: 2022-08-02
SHENZHEN HUADA GENE INST
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  • Application Information

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Problems solved by technology

Among them, the method of embryoid body culture takes a lot of time, the operation process is complicated, and the differentiation efficiency is low.
The method of directional induction of differentiation requires the use of a variety of cytokine combinations, the composition of the differentiation medium is complex, and the cost is high, which is not conducive to large-scale use

Method used

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  • Media and culture methods for ESC differentiation of ectodermal precursor cells
  • Media and culture methods for ESC differentiation of ectodermal precursor cells
  • Media and culture methods for ESC differentiation of ectodermal precursor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] In this example, embryonic stem cells purchased from Harvard University in the United States were used as test materials to conduct differentiation and culture of ectodermal precursor cells. The medium used in this example includes three groups of medium, namely, feeder medium, feeder-free medium and differentiation medium.

[0047] Feeder medium: DMEM / F12 medium plated with mouse fibroblasts, the medium contains 5-30% by weight KOSR, 1×L-Glutamine, 1×MEM-NEAA, 1×2-Mercaptoethanol and 2-10ng / mL of bFGF, the specific dosage of KOSR in this example is 20%, and the dosage of bFGF is 10ng / mL. KOSR, 1×L-Glutamine, 1×MEM-NEAA, 1×2-Mercaptoethanol and bFGF in this example were purchased from GIBCO.

[0048] Feeder-free medium: the base layer is covered with 8-12mg / mL BD Matrigel matrix or 0.2-0.5% fibronectin mTeSR medium or E8 medium. mL of BDMatrigel matrix in mTeSR medium. Among them, BD Matrigel substrate was purchased from BD Company, and mTeSR medium was purchased fro...

Embodiment 2

[0062] In this example, three methods were used to identify the ESC differentiated ectodermal precursor cells obtained in Example 1, namely, QPCR was used to detect the specific gene expression of ectodermal precursor cells, and flow cytometry was used to detect the surface of ectodermal precursor cells. Antigen expression, and immunofluorescence staining to detect ectodermal precursor cell-specific protein expression. Through three detection methods, the cells differentiated from ESCs were further determined to be ectodermal precursor cells, and their differentiation efficiency was quantitatively detected. Details are as follows:

[0063] 1. Three detection methods

[0064] 1.1 QPCR detection of ectodermal precursor cell-specific gene expression

[0065] After the H1 line ESCs were cultured on the differentiation medium for 5 days, the total cellular RNA was extracted, and RNA extraction was performed using the RNA extraction kit purchased from TAKARA. At the same time, th...

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Abstract

The present application discloses a medium and a culture method for ESC differentiation into ectodermal precursor cells. The medium of the present application includes differentiation medium; the differentiation medium is RPMI-1640 medium, DMEM / F12 medium or Knockout DMEM / F12 medium, which contains the first, second and third reagents; the first reagent is the final Activin A at a concentration of 30-60ng / mL or BMP-4 at a concentration of 40-80ng / mL, the second reagent is A83-01 at a final concentration of 1-5μM, and the third reagent is PNU74654 at a final concentration of 1-8μM, 1-10μM Dorsomorphin or 0.5-5% by weight of B-27. The culture medium and culture method of the present application are easy to operate and low in cost, and can quickly obtain ectodermal precursor cells with good growth state and high differentiation efficiency, and the differentiation ratio reaches more than 99%.

Description

technical field [0001] The present application relates to the field of embryonic stem cell differentiation and culture, and in particular, to a medium and a culture method for ESC differentiation into ectodermal precursor cells. Background technique [0002] Embryonic Stem Cell (ESC) is a class of cells with self-renewal and differentiation totipotency isolated from early developing mammalian embryos or primordial germ cells. The cell differentiation of the three germ layers has great application prospects in new drug screening, cell replacement therapy and regenerative medicine research. [0003] Since Thomson et al. successfully established the first human embryonic stem cell line in 1998, ESCs have been favored by researchers due to their excellent proliferation ability and the potential to differentiate into almost all types of cells in the body. Neuroectoderm is the origin of the development of the nervous system and is also an important stage of early mammalian embryo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/079C12N5/0793
CPCC12N5/0618C12N5/0619C12N5/0622C12N2500/84C12N2501/115C12N2500/32C12N2500/44C12N2501/155C12N2501/999C12N2501/998C12N2506/02
Inventor 张家文张曦孙长斌
Owner SHENZHEN HUADA GENE INST
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