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A method for inducing mouse embryonic stem cells to differentiate inner ear hair cells

A technology for the differentiation of inner ear hair cells and stem cells, which can be applied in animal cells, vertebrate cells, artificial cell constructs, etc.

Active Publication Date: 2017-09-12
HANGZHOU S EVANS BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, due to the scarcity of hair cells, research on the molecular mechanism of hair cell development is still very limited

Method used

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  • A method for inducing mouse embryonic stem cells to differentiate inner ear hair cells
  • A method for inducing mouse embryonic stem cells to differentiate inner ear hair cells
  • A method for inducing mouse embryonic stem cells to differentiate inner ear hair cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: The method of combining conditioned medium and factors to induce embryonic stem cells to differentiate into hair cell precursor cells

[0024] Mouse embryonic stem cells ES-D3 (purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were passaged and then placed on a culture plate pre-coated with 1% agar at a mass concentration, and added with conditioned medium at 37°C. , 5%CO 2 Conditioned culture was performed for 4-5 days, and the medium was changed every day. Then transfer the cell mass to a 12-well plate, about 30 cells per well, replace with induction solution I, and store at 37°C, 5% CO 2 Conditional induction culture was carried out for 12-14 days, and the medium was changed every day to obtain hair cell precursor cells differentiated from embryonic stem cells.

[0025] The composition of the final concentration of conditioned medium: volume concentration 40% HepG2 con...

Embodiment 2

[0028] Example 2: Isolation of Chicken Embryo Utriculus Mesenchymal Cells

[0029] Isolate the utricle from fertilized 18-day-old chicken embryos (purchased from Hangzhou Tianyuan Breeding Co., Ltd.), digest with 0.25% trypsin (purchased from Corning Company) at 37°C for 15-20 minutes, and then use DMEM culture medium (add volume final concentration 10% FBS) to stop, pass through a 200-mesh sieve, collect the cell suspension, centrifuge at 1000rpm / min for 6-8 minutes, suspend the cells with DMEM culture medium (added volume final concentration 10% FBS), inoculate in a culture bottle, place at 37 °C, 5% CO 2 Cultivate in an incubator, change the medium for the first time after 48 hours, discard the non-adhered cells, continue to culture the adherent cells to 90% confluence, digest with 0.25% trypsin / EDTA (purchased from Corning Company) and inoculate into cell culture flasks for culture, Recorded as the P1 generation cells, through subculture in this way, until the P3 generati...

Embodiment 3

[0030] Example 3: The combination of factors combined with trophoblast cells induces precursor cells to differentiate into mature inner ear hair cells

[0031] First, the chick embryo utricle mesenchymal cells grown to 90% confluence prepared in Example 2 were soaked in DMEM culture medium containing a final concentration of 2 μg / ml mitomycin C (purchased from Sigma Company), at 37° C. for 5 %CO 2 After culturing in an incubator for 3 hours, the culture medium was discarded, and the cells were washed with PBS buffer solution (purchased from Sangon Biotechnology Co., Ltd.) with a pH value of 7.4. After removing the washing solution, they were pretreated chick embryo utriculus mesenchymal cells.

[0032] Digest the hair cell precursor cells induced and differentiated in Example 1 with accutase (purchased from Gibco Company), let stand for a while to remove undispersed cell clumps, and dilute the cell suspension with 10 5 piece / cm 2 The density was inoculated onto the mesenchym...

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Abstract

The invention discloses a method for inducing the differentiation of mouse embryonic stem cells to obtain inner ear hair cells. Firstly, the conditioned medium and the induction medium are used to induce the embryonic stem cells to differentiate into the hair cell precursor cells; and then the method of combining the induction medium with the trophoblast cells Inducing precursor cells to differentiate into mature inner ear hair cells; the method of the present invention can effectively induce embryonic stem cells to differentiate into inner ear hair cell precursor cells and mature inner ear hair cells, the differentiation rate is high, and the differentiated cells express hair cell-specific genes and proteins, and Possess certain electrophysiological functions of hair cells.

Description

(1) Technical field [0001] The invention relates to a differentiation method of inner ear hair cells, in particular to a method for inducing differentiation of mouse embryonic stem cells to obtain inner ear hair cells. (2) Background technology [0002] Hearing loss is one of the world's chronic diseases that seriously affect the quality of human life, and there is currently no cure. Irreparable damage to hair cells in the mammalian inner ear is considered one of the leading causes of hearing loss. The traditional view is that the auditory hair cells of the human inner ear are specialized terminal cells, which no longer have the ability to regenerate and repair, and their damage is irreversible. In recent years, studies have found that mammalian embryonic or early hair cells may regenerate, and the ectopic occurrence of immature hair cells has been found in adult cochlea by transfecting genes related to hair cell differentiation and development, suggesting that mammalian ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 项黎新潘若浪邵建忠王萍刘小园
Owner HANGZHOU S EVANS BIOSCI LTD
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