Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells

A technology of mesenchymal stem cells and nerve cells, applied in the biological field, can solve the problems of cells that cannot be co-cultured, cell separation, and pathogenic pathogens caused by exogenous pathogens, and achieve the effects of saving time, promoting differentiation, and improving efficiency

Active Publication Date: 2010-12-15
SHANDONG QILU STEM CELL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] However, an inconvenience of this method is that the final cells cannot be effectively separated fro

Method used

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  • Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells
  • Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells
  • Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] (1) Extraction of umbilical cord mesenchymal stem cells

[0051] This example uses the neonatal umbilical cord authorized by the mother as a source of mesenchymal stem cells.

[0052] The neonatal umbilical cord was washed three times in physiological saline containing 1% double antibody to remove the blood stains on the surface, and then cut into small pieces about 1 cm long. Use ophthalmic scissors to cut the umbilical cord longitudinally along the direction parallel to the blood vessels, and peel off the 2 umbilical arteries and 1 umbilical vein from the umbilical cord. Peel off the amniotic membrane on the surface, wash the Huatong glue part with normal saline containing 1% double antibody for 3 times, and cut it into pieces to about 1mm 3 size. Evenly spread the shredded tissue blocks on the 75cm 2 Place in the culture bottle at room temperature for 5-10 minutes to make the tissue pieces stick tightly. Add 5ml of MSC medium. Place at 37°C, 5% CO 2 cultivated ...

Embodiment 2

[0068] (1) Extraction of umbilical cord mesenchymal stem cells

[0069] This example uses the neonatal umbilical cord authorized by the mother as a source of mesenchymal stem cells.

[0070] The neonatal umbilical cord was washed three times in physiological saline containing 1% double antibody to remove the blood stains on the surface, and then cut into small pieces about 1 cm long. Use ophthalmic scissors to cut the umbilical cord longitudinally along the direction parallel to the blood vessels, and peel off the 2 umbilical arteries and 1 umbilical vein from the umbilical cord. Peel off the amniotic membrane on the surface, wash the Huatong glue part with normal saline containing 1% double antibody for 3 times, and cut it into pieces to about 1mm 3 size. Evenly spread the shredded tissue blocks on the 75cm 2 Place in the culture bottle at room temperature for 5-10 minutes to make the tissue pieces stick tightly. Add 5ml of MSC medium. Place at 37°C, 5% CO 2 cultivated ...

Embodiment 3

[0085] (1) Extraction of umbilical cord mesenchymal stem cells

[0086] The extraction steps are the same as in Example 1.

[0087] (2) Preparation of four induction media

[0088] Induction medium The basal medium used was Neurobasal (Invitrogen) supplemented with 2V% B27 (Invitrogen).

[0089] The factors added to the four induction media are as follows.

[0090]The first: 5-azacytidine (50uM, Sigma) and Pam 3 CSK 4 (0.1ug / ml, Invivogen)

[0091] The second: bFGF (200ng / ml, Biovison) and Noggin (1ng / ml, Biovison)

[0092] The third: bFGF (200ng / ml, Biovison), RA (50uM, Sigma), FGF8 (1ng / ml, R&D System) and Wnt3a (1ng / ml, R&D System)

[0093] The fourth type: Bmp4 (200ng / ml, R&D System), Shh (500ng / ml, R&D System), RA (50uM, Sigma), NGF (1ng / ml, R&D System) and BDNF (1ng / ml, R&D System)

[0094] (3) Induced differentiation of umbilical cord mesenchymal stem cells

[0095] In advance, the 24-well cell culture plate was paved with 100ug / ml laminin (Invitrogen) aqueous s...

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Abstract

The invention relates to the field of biotechnology, in particular to an inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells, which comprises the following steps of: cultivating the separated umbilical cord mesenchymal stem cells in an MSC culture medium to spread a whole culture dish; cultivating the stably-subculturing mesenchymal stem cells in the culture dish which is spread with the extracellular matrix by means of the MSC culture medium to be 70% confluence; and leading the 70% confluent mesenchymal stem cells to sequentially pass through four inducing culture mediums to cultivate, wherein the four inducing culture mediums comprise the following factors of 5-azacytidine+Pam3CSK4, bFGF+Noggin, bFGF+RA+FGF8+Wnt3a, and Bmp4+Shh+RA+NGF+BDNF. The inducing method has the benefit effects of being free of chemical substances with cytotoxicity, transgenic technology and nerve cell co-cultivation, and being high in differentiating efficiency and shorter in time consumption.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for inducing differentiation of umbilical cord mesenchymal stem cells into nerve cells. Background technique [0002] Mesenchymal stem cell (MSC) is a type of adult stem cell that is derived from mesoderm, capable of self-renewal, and has the potential to differentiate into three mesoderm lineages: osteoblast, cartilage, and adipose. In recent years, studies have found that MSCs can also transdifferentiate beyond the mesoderm, such as nerve cells and epithelial cells of the ectoderm line, and liver cells and pancreatic cells of the endoderm line. Because MSC has a wide range of sources, is easy to isolate and culture, has low immunogenicity, and does not have the ethical issues faced by embryonic stem cells, this makes its application in regenerative medicine have advantages that other stem cells do not have. Neurodegenerative diseases of the central nervous system, such as...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 范奉群刘东李栋
Owner SHANDONG QILU STEM CELL ENG
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