Method for induced differentiation of inner ear hair cells by virtue of human mesenchymal stem cells

A technology of bone marrow mesenchyme and inner ear hair cells, applied in artificial cell constructs, animal cells, vertebrate cells, etc.

Active Publication Date: 2015-03-04
HANGZHOU S EVANS BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, studies have suggested the possibility of mammalian hair cell regeneration under certain induction c

Method used

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  • Method for induced differentiation of inner ear hair cells by virtue of human mesenchymal stem cells
  • Method for induced differentiation of inner ear hair cells by virtue of human mesenchymal stem cells
  • Method for induced differentiation of inner ear hair cells by virtue of human mesenchymal stem cells

Examples

Experimental program
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Embodiment 1

[0024] Example 1: Isolation of human bone marrow mesenchymal stem cells

[0025] The bone marrow of adult healthy volunteers was washed twice with a pH value of 7.4 phosphate buffered saline (PBS, purchased from Sangon Biotech Co., Ltd.), and the bone marrow was diluted to 10 with PBS. 7 cells / ml of cell suspension, gently added to the upper layer of human lymphocyte separation medium (Ficoll-Hypaque, D = 1.077 ± 0.002g / ml, purchased from Sangon Biotechnology), the volume ratio of the two was 3:2; room temperature (25 ℃), 2200rpm / min after centrifugation for 25 minutes, absorb the middle white cell layer (mononuclear cell layer); add the IMDM basal culture medium (purchased from Corning company), wash the cells, then centrifuge at 1000rpm / min for 6-8 minutes, pour off the culture medium, suspend the cells with the same culture medium, inoculate them in a culture bottle, and place at 37°C, 5% CO 2 Culture in an incubator, change the medium for the first time after 48 hours, di...

Embodiment 2

[0026] Example 2: Isolation of Chicken Embryo Utriculus Mesenchymal Cells

[0027] Utricle was isolated from fertilized 18-day chicken embryo (purchased from Hangzhou Tianyuan Breeding Co., Ltd.), digested with 0.25% trypsin at 37° C. for 15-20 minutes, and added with DMEM culture medium with a final volume concentration of 10% FBS (purchased from Corning Company) 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 solution with a final volume concentration of 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 non-adherent cells, and continue to culture adherent cells to 90% confluence, digest with 0.25% trypsin / EDTA and inoculate into cell culture flasks for culture, which are recorded as P1 generation cells, Through subculture in this way, chicken embryo utriculus mesenchymal cells ...

Embodiment 3

[0028] Example 3: Induction of human bone marrow mesenchymal stem cells in vitro differentiation of inner ear hair cells

[0029] Take the chick embryo utriculus mesenchymal cells grown to 90% confluence prepared in Example 2, soak in DMEM culture medium containing a final concentration of 2 μg / ml mitomycin C (purchased from Sigma Company), 37 ° C, 5% CO 2 After culturing in an incubator for 3 hours, the suspension was discarded and washed with PBS to obtain pretreated chicken embryo utriculus mesenchymal cells.

[0030]Take the P3-P5 generation human bone marrow MSCs obtained by the method in Example 1, and inoculate them into induction solution I, 37°C, 5% CO 2 Induced culture in the incubator for 7 days, in which the medium was changed every 2-3 days, and then changed to induction medium II, 37°C, 5% CO 2 The incubator continued to cultivate for 7-10 days, and the medium was changed every 2-3 days. After digestion with 0.25% trypsin EDTA, the 5 piece / cm 2 The density wa...

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Abstract

The invention discloses a method for induced differentiation of inner ear hair cells by virtue of human mesenchymal stem cells. The method comprises the steps of separating bone marrow MSCs (mesenchymal stem cells) from bone marrow of human as a source, and inducing the bone marrow MSCs by combining factor combination with feed layer cells so as to differentiate the mesenchymal stem cells. According the method, the human mesenchymal stem cells can be induced to be differentiated into the inner ear hair cells, and the cells obtained by differentiation express specific protein of the hair cells and have certain hair cell electrophysiological functions.

Description

(1) Technical field [0001] The invention relates to a method for obtaining inner ear hair cells by inducing differentiation of human bone marrow mesenchymal stem cells. (2) Background technology [0002] Irreparable damage to hair cells in the mammalian inner ear is one of the leading causes of hearing loss. It is generally believed that the auditory hair cells of the human inner ear do not have the ability to self-regenerate and repair, and their damage is irreversible. In recent years, studies have suggested the possibility of mammalian hair cell regeneration under certain induction conditions, but there are still great challenges in the regeneration and repair of hair cells in the process of disease. Therefore, finding the cell source of hair cell regeneration and developing intervention strategies for repairing hair cell damage are the key to the treatment of hearing loss. [0003] Stem cells have become a research hotspot in the field of tissue regeneration and repair...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 邵建忠潘若浪项黎新刘小园王萍
Owner HANGZHOU S EVANS BIOSCI LTD
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