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Method for inducing human mesenchymal stem cells differentiation to oligoden drocyte

A technology of oligodendrocytes and bone marrow mesenchyme, applied in biochemical equipment and methods, animal cells, nervous system cells, etc., can solve the problems of complicated separation process, non-autologous transplantation, and high price

Inactive Publication Date: 2009-06-10
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The current methods to obtain oligodendrocytes or oligodendrocyte precursor cells are: 1) It can be obtained from embryonic or adult brain tissue and cultured in vitro, but it is difficult to obtain enough donor cells, and there is allogeneic rejection
2) It is also possible to obtain the desired cell type from various types of stem cells with pluripotency, such as embryonic stem cells and neural stem cells, through in vitro culture induction differentiation, but the main problem is the oligodendrocytes derived from embryonic stem cells or neural stem cells, 3) Oligodendrocytes derived from autologous MSCs, the traditional method of separating MSCs is to use magnetic beads, which is expensive, the separation process is complicated, easy to pollute, and growth factors induce differentiation reactions. Tumorogenic; currently there is no simple and reliable method to selectively differentiate from human bone marrow mesenchymal stem cells (hBMSCs) into oligodendrocytes Differentiation to oligodendrocytes is a prerequisite for cell transplantation to treat neurological injuries such as multiple sclerosis and demyelinating diseases
In vitro experiments also proved that bone marrow-derived stem cells could express markers of neurons, astrocytes and Schwann cells through induction, but oligodendrocyte markers were less expressed

Method used

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  • Method for inducing human mesenchymal stem cells differentiation to oligoden drocyte
  • Method for inducing human mesenchymal stem cells differentiation to oligoden drocyte
  • Method for inducing human mesenchymal stem cells differentiation to oligoden drocyte

Examples

Experimental program
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Effect test

Embodiment 1

[0013] Example 1 In vitro culture and expansion of human bone marrow mesenchymal stem cells

[0014] hBMSCs (≥70%) were derived from Cambrex, and samples were collected from 38-year-old healthy male youth donors. Primary hBMSCs were inoculated in DMEM medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator. After 48 hours, rinse twice with PBS to remove unattached cells and replace with new culture medium. When the adherent cells were 90% confluent, they were digested with 0.25% trypsin and subcultured at a ratio of 1:3 to continue to expand and culture.

[0015] The morphological characteristics of the hBMSCs obtained by this method: when the cells are just cultured, a large number of round cells are suspended in the culture dish, and these unattached blood cells can be removed by changing the medium, and the adherent cells are hBMSCs. The initially inoculated hBMSCs are round, large in size, translucent, and strong in refraction; 24 hours after in...

Embodiment 2

[0016] Example 2 Using Flow Cytometry to Analyze the Characteristics of Bone Marrow Mesenchymal Stem Cells

[0017] When the cells grew to 90% confluence, the cells were collected for flow cytometry detection, and the adherent hMSCs in the plastic dish were digested into single cells with 0.25% EDTA-containing trypsin, washed three times with PBS, counted and adjusted To an appropriate concentration (1×106 / ml), fixed with alcohol, washed three times with PBS; treated with TritonX-100 and hydrogen peroxide; added normal goat serum to block at room temperature for 30 minutes; added CD34, CD45, or CD90 antibodies respectively for 2 hours at room temperature ; Add FITC fluorescent antibody to incubate together, and then use FACS to analyze the number of CD90 positive cells.

[0018] The results showed that flow cytometry analysis was performed when the adherent cells were nearly 90% confluent. The results showed that CD90(+) cells reached 83.8% to 97.7%, with an average of 94.78+...

Embodiment 3

[0019] Example 3 Induction of human bone marrow mesenchymal stem cells to differentiate into oligodendrocytes in vitro and phenotype identification refer to Dezawa et al. (Dezawa M, Takahashi I, Esaki M, et al. Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur J Neurosci, 2001, 14: 1771-1776) method of chemically inducing hBMSCs: cells close to confluence were pre-induced with 1 mM β-mercaptoethanol (β-ME) DMEM for 24 hours; 10% fetal bovine serum DMEM induction solution of RA (RA) for 3 days; then the cells were divided into MFS group (positive control group) and F3 group and continued to culture for 3 days. 10ng / ml basic fibroblast growth factor (bFGF), 5ng / ml platelet growth factor (PDGF), 200ng / mL heregulin (HRG) DMEM medium; F3 group cells were cultured in DMEM containing 10% fetal bovine serum, 20nM F3 base.

[0020] The characteristics of bone marrow mesenchymal stem cells induced into oligodendrocyt...

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Abstract

The invention discloses a method for inducing differentiation from human mesenchymal stem cells to oligodendrocyte. The invention aims to provide a method for obtaining a large quantity of oligodendrocyte and using the oligodendrocyte to treat diseases such as myelin sheath damage or demyelination. The method is to obtain a large quantity of the autogenous human mesenchymal stem cells by a simple platy adhesion method, and to use adhesion molecules F3 / Contactin to induce differentiation from the human mesenchymal stem cells to the oligodendrocyte. The method adopts an easy, simple and convenient means to perform in vitro regulation on differentiation from the human mesenchymal stem cells to the oligodendrocyte, and achieves the aims of acquisition of the autogenous oligodendrocyte, preparation of a large quantity of the autogenous oligodendrocyte, and low cost. The method realizes the aim of applying the oligodendrocyte from the autogenous human mesenchymal stem cells to treat the demyelination disease, and has better economic benefit and social benefit.

Description

technical field [0001] The invention relates to a method for regulating cell-induced differentiation, in particular to a new method for regulating the differentiation of human bone marrow mesenchymal stem cells cultured in vitro into oligodendrocytes. Background technique [0002] Oligodendrocytes are the myelin-forming cells of the central nervous system, which surround the axons of nerve fibers and form insulating myelin sheaths. Therefore, the formation of myelin sheaths in the central nervous system of vertebrates is very important for the growth, development, and development of neuron axons. The transmission of nerve signals plays a crucial role. When myelin is damaged, either by destruction of the structurally intact myelin sheath—demyelination, as in trauma, multiple sclerosis (MS), or dysmyelination due to abnormal oligodendrocyte development (dysmyelination), such as congenital cerebellar disease, can cause serious central nervous system lesions. The lack of oligo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/079
Inventor 朱玲玲肖志诚范明陆莉赵彤
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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