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Method for directionally differentiating induced multipotent stem cells into inner ear hair cell-like cells

A technology of inner ear hair cells and pluripotent stem cells, applied in the field of biomedicine

Active Publication Date: 2021-05-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, significant challenges remain regarding the efficiency and safety of inner ear hair cell regeneration in vivo.

Method used

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  • Method for directionally differentiating induced multipotent stem cells into inner ear hair cell-like cells
  • Method for directionally differentiating induced multipotent stem cells into inner ear hair cell-like cells
  • Method for directionally differentiating induced multipotent stem cells into inner ear hair cell-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Directed differentiation of induced pluripotent stem cells into inner ear progenitor cells

[0034] (1) Induced pluripotent stem cells with good growth were cultured normally, digested into single cells with Accutase, diluted with DMEM / F12 (1:1) medium, centrifuged at 300g for 5min, resuspended and counted. According to 5μg / cm 2Laminin diluted with PBS was added to a 12-well plate, and the solution was discarded after standing at 37°C for 2 hours. Laminin-coated 12-well plates were inoculated with 1×10 4 The cells were cultured with the progenitor cell medium supplemented with Y-27632 at a final concentration of 10 μmol / L for 2 days, and then the medium was changed every other day for co-culture for 12 days. The progenitor cell medium is 1% N2supplement, 2% B27 supplement, 0.2% Normocin (500×) and 1mmol / L acetone added to the DMEM / F12 (1:1) medium by volume sodium nitrate, FGF3 at 50ng / mL, FGF10 at 50ng / mL.

[0035] (2) Extract the total RNA from inner ear...

Embodiment 2

[0037] Example 2 Directed differentiation of inner ear progenitor cells into inner ear hair cell-like cells

[0038] (1) Take eggs fertilized for 18.5 days, take out the chicken embryo in a sterile environment, separate the utricle of the chicken embryo through stereoscopic dissection, digest the utricle with thermophilic protease solution for 30 minutes, and then digest it with 0.25% trypsin after the digestion is terminated After 5 minutes, the digestion was stopped, and the mixture was removed with a 200-mesh sieve, centrifuged at 300g for 5 minutes, and the cells were resuspended in the growth medium and inoculated into a single culture dish, which was recorded as P0 generation cells. P0 generation chicken embryo utricle cells were cultured until the confluence reached 90%, and P1 generation cells were obtained. Repeated culture and passage until P3 generation cells were obtained. Chicken embryo utricle cells were added with mitomycin at a final concentration of 2 μg / mL C ...

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Abstract

The invention provides a method for directionally differentiating induced multipotent stem cells into inner ear hair cell-like cells. A culture medium with definite chemical components is mainly utilized, small molecule compounds are added at proper time to induce and differentiate iPSC into inner ear progenitor cells, and then the inner ear progenitor cells and chick embryo elliptical capsule cells are co-cultured, so that the inner ear hair cell-like cells are obtained. According to the method, the induced multipotent stem cells can be effectively and directionally differentiated into the inner ear hair cell-like cells, and the inner ear hair cell-like cells obtained through differentiation have cilia bundle structures, express inner ear hair cell specific proteins and have hair cell electro-physiological functions.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for directional differentiation of induced pluripotent stem cells into inner ear hair cell-like cells. Background technique [0002] Hearing impairment is a major global public health problem. According to estimates by the World Health Organization in 2019, there are about 460 million people worldwide with hearing impairment (http: / / www.who.int / en / ). Inner ear hair cells are an important type of sensory epithelial cells in the human hearing system. They convert the sound waves felt by the inner ear into electrical stimulation through mechanical-electrical conversion, and conduct them to the brain along the auditory nerve to form hearing. It is generally believed that mammalian inner ear hair cells are difficult to regenerate after damage, so inner ear hair cell damage is an important cause of human hearing loss. Recent studies have suggested that under certain experimental co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0627C12N5/062C12N2533/52C12N2533/54C12N2506/45C12N2500/80C12N2500/38C12N2500/30C12N2501/11
Inventor 管敏鑫陈潮冀延春孟飞龙
Owner ZHEJIANG UNIV
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