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Method for inducing and acclimating epidermal stem cells into muscle cells

A technology of epidermal stem cells and muscle cells, which is applied in the field of using epidermal stem cells to induce domestication into muscle cells, and can solve the problems of limited application range of epidermal stem cells

Inactive Publication Date: 2010-06-30
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The scope of application of epidermal stem cells is also limited by the influence of unipotent cells

Method used

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  • Method for inducing and acclimating epidermal stem cells into muscle cells
  • Method for inducing and acclimating epidermal stem cells into muscle cells
  • Method for inducing and acclimating epidermal stem cells into muscle cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of epidermal stem cells before induction and domestication

[0039] 1. Preparation of epidermal stem cells:

[0040] Direct source and original source: With the consent of the patient, it was taken from healthy circumcision patients in the 301 Hospital Surgical Clinic.

[0041] (1) Take fresh adult foreskin (isolated for about 1 to 4 hours, stored at 4°C);

[0042] (2) Soak the foreskin in a dilute iodophor solution (containing 20% ​​iodophor) for 1 min;

[0043] (3) PBS / physiological saline rinse 3 times to wash away blood clots (to prevent affecting enzyme digestion);

[0044] (4) Trim the foreskin, remove the fat tissue, and then rinse with PBS / saline several times, and finally cut into 1cm 2 The left and right organizational blocks;

[0045] (5) Put the tissue block in a 6cm dish containing a solution of neutral protease (3mg / ml; D-hanks as solvent) and treat it at 4°C for 14-16h. If the treatment is not enough, place it in a 37°C incubator (or directly P...

Embodiment 2

[0119] Example 2 Directed induction and domestication of epidermal stem cells into muscle cells

[0120] 1. Medium composition

[0121] (1) The composition and dosage of muscle induction medium are: 90% IMDM (Bonate); 1uM Dexamethasone (sigma D1756); 0.2mM indomethacin (sigma, cat.no.17378); 0.5mMinsulin (sigma, cat.no. 19278); 0.5mM isobutyl-methylxanthine (sigma, cat. no. 17018); 10% FBS (Gibco, cat. no. 10437010).

[0122] (2) The components and dosage of dry maintenance medium are: 98% Epilife (Cascade Biologics, cat.no.M-EPI-500-CA), 1% PS double antibody (double antibody Invitrogen, cat.no.15070) And 1% HKGS additive (HKGS, Cascade Biologics, cat.no.S-001-5).

[0123] 2. Induction and domestication method:

[0124] (1) Pre-treatment for domestication induction:

[0125] Wash the cells with PBS 2-3 times before the start of cell induction. The purpose is to determine the number of small round cells and change the fresh pilife medium. After changing the medium, culture for more tha...

Embodiment 3

[0129] Example 3 Identification and result analysis of target cells

[0130] 1. Immunofluorescence identification of induced differentiation results

[0131] 1. Identification method: In the experimental verification of induction into muscle cells, the identification method of muscle cell marker (a-SMA) detected by immunofluorescence is as follows:

[0132] (1) Take the cells one, two and three weeks after induction, wash the cells twice with PBS, and fix them with 1ml of 4% paraformaldehyde for 30 minutes

[0133] (2) Discard the fixative, wash the cells with PBS 3 times, 5 minutes each

[0134] (3) Add 1ml of 0.05% permeabilization solution for 30 minutes

[0135] (4) Discard the permeabilization solution and wash the cells 3 times with PBS for 5 minutes each time

[0136] (5) Add 500ul sheep serum blocking solution for 1 hour. Dilute the primary antibody according to the required ratio

[0137] (6) Add the diluted SMA primary antibody, put it in a humidified box, and refrigerate overni...

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Abstract

The invention discloses a method for inducing and acclimating epidermal stem cells into muscle cells, comprising the following steps of: mixing an eplife culture medium and an induction culture medium according to different proportions, and ensuring that the epidermal stem cells have enough time fit for the continuous reduction of the eplife culture medium and the gradual increase of the induction culture medium, which is beneficial to reducing the differentiation to the epidermis and increasing the transformational probability to targeted cells. The method is simple and very effective to directively induce and acclimate the epidermal stem cells into the muscle cells, overcomes the prejudice from technical personnel in the filed, subverts a judgment that the epidermal stem cells are unipotent stem cells for the first time, and proves that the epidermal stem cells have the multi-directional differentiation potential.

Description

Technical field [0001] The invention relates to a method for inducing cells, in particular to a method for inducing and domesticating into muscle cells by using epidermal stem cells. Background technique [0002] Human research on epidermal stem cells has a history of decades, but because of their difficulty in inducing differentiation, they have been considered unipotent stem cells, that is, they can only differentiate into relevant components of the epidermis. Regenerative medicine is very popular in the field of medicine. The research of stem cells has occupied an important low position in the field of regenerative medicine. Because of their multi-differentiation potential, stem cells can differentiate into various tissues and organs under specific conditions, which has attracted the attention of scientists. . Embryonic stem cells are totipotent stem cells that can differentiate into all tissues and organs except the placenta. However, since the research of embryonic stem ce...

Claims

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Application Information

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IPC IPC(8): C12N5/08
Inventor 韩为东付小兵陈美霞
Owner GENERAL HOSPITAL OF PLA
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